Compositions and Methods to Regulate Renalase in the Treatment of Diseases and Disorders

ABSTRACT

The invention provides compositions and methods for binding and inhibiting renalase. In one embodiment, the renalase binding molecule inhibits renalase activity. Thus, in diseases and conditions where a reduction of renalase activity is beneficial, such inhibitory renalase binding molecules act as therapeutics.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.RC1DK086465, RC1DK086402, DK54021 and R01DK081037 awarded by theNational Institutes of Health.

The Government has certain rights in this invention

BACKGROUND OF THE INVENTION

Renalase (RNLS) is a protein produced predominantly in the kidney,heart, skeletal muscle, testes and to a lesser extent in other tissues(Xu et al., 2005 J Clin Invest. 115 (5):1275-80 and Wang et al., 2008Mol Biol Rep. 35(4):613-20). Two isoform variants of renalase have beendescribed, Renalase-1 and Renalase-2. These two forms of renalase differdue to differential splicing of the final exon. Renalase has beendescribed as a novel flavin adenine dinucleotide-containing monoamineoxidase with an activity that selectively deaminates the catecholaminesepinephrine, norepinephrine and dopamine. A deficiency of renalase inthe plasma of patients with end-stage renal disease, in comparasion tohealthy individuals, has been described. Catecholamines play a majorrole in the maintenance and modulation of blood pressure, including indisease, through effects on cardiac output and vascular resistance. Theinfusion of a recombinant form of renalase into rats caused a decreasein cardiac contractility, heart rate, and blood pressure. Patients withrenal failure have been characterized with heightened levels ofcirculating catecholamines which correlate with hypertension and greatermortality through cardiovascular complications. Thus the proteinrenalase may play a role in the control and maintenance ofcatecholamine-induced changes in blood pressure and the deficiency ofrenalase observed in renal disease patients may be detrimental tooutcomes.

A deficiency of renalase in the plasma of patients with end-stage renaldisease, in comparasion to healthy individuals, has been described.Patients with renal failure have been characterized with heightenedlevels of circulating catecholamines which correlate with hypertensionand greater mortality through cardiovascular complications. Thus theprotein renalase may play a role in the control and maintenance ofcatecholamine-induced changes in blood pressure and the deficiency ofrenalase observed in renal disease patients may be detrimental tooutcomes. However, little is know about the role of renalase in cancer.

An essential feature of cancer is dysregulation of cell senescence anddeath. Renalase (RNLS) is a secreted flavo-protein that protects againstischemic and toxic cellular injury by signaling through the plasmamembrane calcium ATPase PMCA4b to activate the PI3K/AKT, and MAPKpathways.

Skin cancer is a common human malignancy, and its incidence has beenincreasing in developed countries (Gray-Schopfer et al., 2007 Nature.445:851-7; Lowe et al., 2014 Mayo Clinic Proceedings. 89:52-9; Lesinskiet al., 2013 Future oncology. 9:925-7). Melanoma is the deadliest formof skin cancer, with low survival rates once it becomes unresectable(Lowe et al., 2014 Mayo Clinic Proceedings. 89:52-9). It is amolecularly heterogeneous disease and some of the key alterations insignaling pathways that participate in disease development andprogression have been identified. The Ras/Raf/MEK/ERK and the PI3K/AKTsignaling pathways play key roles in the pathogenesis of melanoma(Gray-Schopfer et al., 2007 Nature. 445:851-7; Lesinski et al., 2013Future oncology. 9:925-7; Yajima et al., 2012 Dermatology research andpractice. 2012:354191). Mutations in Ras, Raf, PI3K or PTEN (PI3Kinhibitor) can lead to the sustained activation of ERK and AKT, which inturn promote cell survival and proliferation. Dankort et al.demonstrated this well with conditional melanocyte-specific expressionof BRaf^(V600E) in mice, none of whom developed melanoma, however,revealed 100% penetrance of melanoma development when combined withsilencing of the Pten tumor suppressor gene (Dankort et al., 2009 Naturegenetics. 41:544-52). The elucidation of these pathogenic pathways hasfacilitated the development of specific inhibitors that targethyper-activated kinases. While these agents have proven effective in thetreatment of selective groups of patients with metastatic melanoma,their beneficial actions are often short lived, hence the pressing needfor the identification of additional therapeutic targets.

RNLS expression is markedly increased in melanoma tumors, andspecifically in CD163+ tumor associated macrophages (TAMs). In a cohortof patients with primary melanoma, disease-specific survival wasinversely correlated with RNLS expression in the tumor mass, suggestinga pathogenic role for RNLS. Inhibition of RNLS signaling using siRNA,anti-RNLS antibodies, or a RNLS derived inhibitory peptide significantlydecreases melanoma cells survival in vitro. Anti-RNLS therapy with amonoclonal antibody markedly inhibits melanoma tumor growth in axenograft mouse model. Treatment with m28-RNLS (also known as 1D-28-4),caused a marked reduction in endogenous RNLS expression, and in totaland phosphorylated STAT3 in CD163⁺ TAMs. Increased apoptosis in tumorcells was temporally related to p38 MAPK mediated activation of theB-cell lymphoma 2 related protein Bax. Expression of the cell cycleinhibitor p21 increased and cell cycle arrest was documented. Theseresults indicate that increased RNLS production by CD163⁺ TAMsfacilitates melanoma growth by activating STAT3, and that inhibition ofRNLS signaling has potential therapeutic application in the managementof melanoma.

Improved methods for the detection of renalase in bodily fluids andtissues may aid in the diagnosis and prognosis of renal disease,cardiovascular disease and/or cancer. However, the validation ofrenalase as a relevant biomarker requires highly selective reagents forits detection. Antibody-based technologies are widely used for thedetection of biomarkers. To date there have been only a small number ofreagent antibodies raised against renalase with no to minimalcharacterization.

Pancreatic cancer is one of the most lethal neoplasms, causingapproximately 330,000 death globally and 40,000 in the US (World CancerReport 2014. WHO Press; 2014). Pancreas cancer is difficult to detect,and most cases are diagnosed at a late stage (Nolen et al., 2014 PLoSONE. 9(4):e94928). Although there has been some progress in the use ofchemotherapy of this cancer, the disease remains extremely resistant toall drugs therapies (Hidalgo et al., 2010 New England Journal ofMedicine. 362(17):1605-17). The overall 5 year survival for individualswith pancreatic cancer is <5% (Hidalgo et al., 2010 New England Journalof Medicine. 362(17):1605-17), and additional therapeutic targets areneeded.

The development of pancreatic cancer relies on the stepwise accumulationof gene mutations (Jones et al., 2008 Science. 321(5897):1801-6), someof which cause abnormal MAPK, PI3K and JAK-STAT signaling. Progressionfrom minimally dysplastic epithelium to dysplasia to invasive carcinomareflects the stepwise accumulation of gene mutations that eitheractivate oncogenes (e.g. KRAS2), or inactivate tumor suppressor genes9e.g. CDKN2a/INK4a, TP53 and DPC4/SMaD4) (Hidalgo et al., 2012 Annals ofOncology. 23(suppl 10):x135-x8). Ninety-five, 90 and 75% of pancreatictumors carry mutations in KRAS2, CDKN2a, and TP53, respectively. Thesemutations result in sustained and dysregulated proliferation thatcharacterizes cancer growth. The mutational landscape and core signalingpathways in pancreatic ductal adenocarcinoma (PDAC) have been definedthrough a comprehensive genetic analysis of 24 advanced PDACs (Jones etal., 2008 Science. 321(5897):1801-6). These data indicate that mostPDACs contain a large number of genetic changes that are primarily pointmutations, and which affect approximately 12 cell signaling pathways.

That study also identified five hundred and forty one genesoverexpressed in PDAC by at least 10 fold in 90% of the tumors. Thisincluded a 2 to 4 fold increase in the recently characterized protein,renalase (RNLS), in tumors or in tumor derived cell lines. RNLS, a novelsecreted flavo-protein (Xu et al., 2005 J Clin Invest. 115(5):1275-80;Desir et al., 2012 J Am Heart Assoc. 1(e002634; Desir et al., 2012 J AmSoc Hypertens. 6(6):417-26; Li et al., 2008 Circulation.117(10):1277-82) with NADH oxidase activity, (Farzaneh-Far et al., 2010PLoS One. 5(10):e13496; Beaupre et al., 2015 Biochemistry.54(3):795-806) promotes cell and organ survival (Lee et al., 2013 J AmSoc Nephrol. 24(3):445-55) through a receptor-mediated process that isindependent of its intrinsic enzymatic activities (Wang et al., 2014Journal of the American Society of Nephrology.DOI:10.1681/asn.2013060665). RNLS rapidly activates protein kinase B(AKT), the extracellular signal-regulated kinase (ERK), and the mitogenactivated protein kinase (p38). Chemical inhibition of either ERK or AKTabrogated the protective effect of RNLS (Wang et al., 2014 Journal ofthe American Society of Nephrology. DOI:10.1681/asn.2013060665).

Accordingly, there exists a need for improved methods and compositionsthat bind renalase, such as antibodies, for the detection, diagnosis,prevention and treatment of diseases or disorders including renaldisease, cardiovascular disease, and cancer. The present meets thisneed.

SUMMARY

The invention includes compositions comprising a renalase inhibitor,which may be a chemical compound, a protein, a peptide, apeptidomemetic, a renalase receptor, a renalase receptor fragment, anantibody, an antibody fragment, an antibody mimetic, a ribozyme, a smallmolecule chemical compound, an short hairpin RNA, an antisense nucleicacid molecule, siRNA, miRNA, a nucleic acid encoding an antisensenucleic acid molecule, a nucleic acid sequence encoding a protein. Insome embodiments, the renalase inhibitor is a renalase binding molecule.In some embodiments, the renalase binding molecule is an antibody orbinding portion thereof. In some embodiments, the renalase bindingmolecule that specifically binds to renalase with an affinity of atleast 10⁻⁶M. In some embodiments, the renalase binding moleculespecifically binds a peptide sequence selected from the group consistingof SEQ ID NO: 1-7. In some embodiments, the renalase is human renalase.In various embodiments, the antibody may be a monoclonal antibody, apolyclonal antibody, a single chain antibody, an immunoconjugate, adefucosylated antibody, and a bispecific antibody. In some embodiments,the immunoconjugate comprises a therapeutic agent or a detection moiety.In various embodiments, the antibody may be a humanized antibody, achimeric antibody, a fully human antibody, an antibody mimetic. In oneembodiment, the antibody comprises at least one selected from the groupconsisting of: a) the heavy chain CDR1 sequence selected from the groupconsisting of SEQ ID NO: 11 and SEQ ID NO: 19; b) the heavy chain CDR2sequence selected from the group consisting of SEQ ID NO: 12 and SEQ IDNO: 20; c) the heavy chain CDR3 sequence selected from the groupconsisting of SEQ ID NO: 13 and SEQ ID NO: 21; d) the light chain CDR1sequence selected from the group consisting of SEQ ID NO: 14 and SEQ IDNO: 22; e) the light chain CDR2 sequence selected from the groupconsisting of SEQ ID NO: 15 and SEQ ID NO: 23; f) the light chain CDR3sequence selected from the group consisting of SEQ ID NO: 16 and SEQ IDNO: 24. In some embodiments, the antibody specifically binds apolypeptide comprising the amino acid sequence of SEQ ID NO: 4. In someembodiments, the antibody comprises at least one selected from the groupconsisting of: a) the heavy chain CDR1 sequence selected from the groupconsisting of SEQ ID NO: 27 and SEQ ID NO: 35; b) the heavy chain CDR2sequence selected from the group consisting of SEQ ID NO: 28 and SEQ IDNO: 36; c) the heavy chain CDR3 sequence selected from the groupconsisting of SEQ ID NO: 29 and SEQ ID NO: 37; d) the light chain CDR1sequence selected from the group consisting of SEQ ID NO: 30 and SEQ IDNO: 38; e) the light chain CDR2 sequence selected from the groupconsisting of SEQ ID NO: 31 and SEQ ID NO: 39; f) the light chain CDR3sequence selected from the group consisting of SEQ ID NO: 32 and SEQ IDNO: 40. In some embodiments, the antibody specifically binds apolypeptide comprising the amino acid sequence of SEQ ID NO: 6. In someembodiments, the antibody comprises at least one selected from the groupconsisting of: a) the heavy chain CDR1 sequence SEQ ID NO: 43; b) theheavy chain CDR2 sequence SEQ ID NO: 44; c) the heavy chain CDR3sequence SEQ ID NO: 45; d) the light chain CDR1 sequence SEQ ID NO: 46;e) the light chain CDR2 sequence SEQ ID NO: 47; f) the light chain CDR3sequence SEQ ID NO: 48. In some embodiments, the antibody specificallybinds a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.In some embodiments, the antibody comprises a heavy chain sequenceselected from the group consisting of SEQ ID NOs: 9, 17, 25, 33, and 41.In some embodiments, the antibody comprises a light chain sequenceselected from the group consisting of SEQ ID NOs: 10, 18, 26, 34, and42. In embodiment, the invention is a composition comprising an antibodythat binds to renalase and competes with the binding of the antibody ofclaim 3 to renalase.

In another embodiment, the invention is a method of treating orpreventing a disease or disorder associated with renalase in a subject,including the step of administering to the subject at least one renalaseinhibitor. In some embodiments, the renalase inhibitor is administeredto the subject in combination with a second therapeutic agent. In someembodiments, the disease or disorder associated with renalase isselected from the group consisting of renal disease, cardiovasculardisease, cancer, and any combination thereof. In some embodiments, wherethe disease or disorder is cancer, the cancer is pancreatic cancer ormelanoma.

In another embodiment, the invention is an isolated nucleic acidmolecule encoding a renalase binding molecule, such as, but not limitedto, an antibody. In another embodiment, the invention is an expressionvector comprising a nucleic acid molecule encoding a renalase bindingmolecule, such as, but not limited to, an antibody. In anotherembodiment, the invention is a host cell comprising a nucleic acidmolecule encoding a renalase binding molecule, such as, but not limitedto, an antibody.

In another embodiment, the invention is a method of diagnosing a diseaseor disorder in a subject in need thereof, the method including the stepsof determining the level of renalase in a biological sample of thesubject, comparing the level of renalase in the biological sample of thesubject with a comparator control, and diagnosing the subject with adisease or disorder when the level of renalase in the biological sampleof subject is elevated when compared with the level of renalase of thecomparator control. In one embodiment, the method includes theadditional step of administering a treatment to the subject that wasdiagnosed as having a disease or disorder. In one embodiment, the levelof renalase in the biological sample is determined by measuring thelevel of renalase mRNA in the biological sample. In one embodiment, thelevel of renalase in the biological sample is determined by measuringthe level of renalase polypeptide in the biological sample. In oneembodiment, the level of renalase polypeptide in the biological sampleis determined using a renalase binding molecule. In one embodiment, thelevel of renalase in the biological sample is determined by measuring anactivity (e.g., enzymatic activity, substrate binding activity, receptorbinding activity, etc.) of renalase polypeptide in the biologicalsample. In one embodiment, the level of renalase in the biologicalsample is determined to be elevated when the level of renalase isincreased by at least 10%, by at least 20%, by at least 30%, by at least40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%,by at least 90%, by at least 100%, by at least 200%, by at least 300%,by at least 400%, by at least 500%, by at least 600%, by at least 700%,by at least 800%, by at least 900%, by at least 1000%, when comparedwith a comparator control. In various embodiments, the comparatorcontrol is at least one selected from the group consisting of: apositive control, a negative control, a historical control, a historicalnorm, or the level of a reference molecule in the biological sample. Inone embodiment, the disease or disorder is at least one selected fromthe group consisting renal disease, cardiovascular disease, cancer, andany combination thereof. In one embodiment, when the disease or disorderis cancer, and the cancer is pancreatic cancer or melanoma. In oneembodiment, the subject is human.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the invention, will be better understood whenread in conjunction with the appended drawings. For the purpose ofillustrating the invention, there are shown in the drawings embodimentswhich are presently preferred. It should be understood, however, thatthe invention is not limited to the precise arrangements andinstrumentalities of the embodiments shown in the drawings.

FIG. 1, comprising FIGS. 1A and 1B, is a series of images showing timecourse of renalase-dependent cell signaling. FIG. 1A shows humanembryonic kidney cells (HK-2) incubated with renalase and activation ofprotein kinase B (AKT) and extracellular-signal regulated kinase (ERK)determined by Western blot analysis; representative blot is shown,(n=3); signals normalized to glyceraldehyde 3-phosphate dehydrogenaseloading control (n=3); changes over baseline statistically significantat from 1 to 60 minutes for ERK, and AKT (T308) and at 30 minutes onlyfor AKT (S473). FIG. 1B shows that renalase upregulates theanti-apoptotic molecule Bcl-2 in HK-2 cells and human umbilical veinendothelial cells (HUVEC).

FIG. 2 is an image showing renalase isoforms Ren1-7: exons numbered from1 to 10; RP-224, renalase peptide amino acid 224 233 of Ren1 or Ren2;RP-220, amino acids 220-239; RP-H220, histidine-tagged RP-220;RP-Scr220, scrambled RP-220.

FIG. 3 is a chart showing that ERK or AKT inhibition abrogatesprotective effect of renalase peptide: WT mice subjected to sham surgeryor to 30 minutes of renal ischemia and reperfusion; RP-H220 or vehicle(saline) injected 10 minutes before renal ischemia. ERK inhibitorPD98059 or the PI3K/AKT inhibitor wortmannin abrogated RP-H220'sprotective effect.

FIG. 4 is an image showing sequence alignments of the peptides in Table1 and where these peptides correspond to the renalase-1 or 2 sequences.

FIG. 5, comprising FIGS. 5A through 5J, is a series of images showingsequences of antibodies that bind to renalase; complementaritydetermining regions (CDR) are underlined. FIGS. 5A and 5B show thesequences for 1D-28-4 heavy chain and light chain coding sequences,respectively. FIGS. 5C and 5D show the sequences for 1D-37-10 heavychain and light chain coding sequences, respectively. FIGS. 5E and 5Fshow the sequences for 1F-26-1 heavy chain and light chain codingsequences, respectively. FIGS. 5G and 5H show the sequences for 1F-42-7heavy chain and light chain coding sequences, respectively. FIGS. 5I and5J show the sequences for 3A-5-2 heavy chain and light chain codingsequences, respectively.

FIG. 6 is a chart showing renalase expression in cancer cell lines:expression determined by quantitative PCR and normalized to actinexpression.

FIG. 7 is an image showing renalase expression in melanocytes: markedincreased in renalase expression in nevus and melanoma compared tonormal skin.

FIG. 8 is a chart showing that anti-renalase monoclonal inhibits A375.S2melanoma cells in culture and shows synergism with temozolamide: Cellviability measured by the WST-1 methods at 72 hrs post treatment;RenAb-10: renalase monoclonal, 10 μ/m1; TMZ: temozolamide, 100 or 150μg/ml.

FIG. 9 is a chart showing that anti-renalase monoclonal inhibits A375.S2melanoma cells in culture and shows synergism with dacarbazine: Cellviability measured by the WST-1 methods at 72 hrs post treatment;RNLSMono: renalase monoclonal.

FIG. 10 is a chart showing that anti-renalase monoclonal inhibitsSk-Mel-28 melanoma cells in culture and shows synergism withtemozolamide: Cell viability measured by the WST-1 methods at 72 hrspost treatment; RenAb-10: renalase monoclonal, 10 μ/ml, TMZ100:temozolamide, 100 μg/ml.

FIG. 11 is a chart showing that anti-renalase monoclonal inhibitsleukemic cell line in culture: CCL-119 cells in culture treated withantirenalase monoclonal antibody for 24 hours; cell survival measured bythe WST-1 method (n=3, *P,0.05).

FIG. 12 is a chart showing that anti-renalase polyclonal inhibitspancreatic cancer cell line MiaPac.

FIG. 13 is a chart showing anti-renalase monoclonal inhibits pancreaticcancer cell line Panc1.

FIG. 14 is a photomicrograph comparing melanoma cells in culture withand without a renalase monoclonal. The renalase antibody markedlydecreases the number of live cells.

FIG. 15 is a chart demonstrating that renalase monoclonal antibodies1C-22-1 and 1D-37-10 inhibit melanoma cells in culture.

FIG. 16 is a series of images showing increased mortality in patientwith melanomas expressing high renalase levels: renalase expressionmeasured by AQUA in biopsy specimens from 263 patients with melanomas;tumor mask obtained using antibodies against S-100 and gp100; Follow upperiod on x axis in months; % cumulative survival shown on Y axis.

FIG. 17, comprising FIGS. 17A through 17D, is a series of images andcharts showing RNLS overexpression in melanoma, and association withpoor patient outcome. FIG. 17A is an image showing RNLS expressiondetected using anti-RNLS-m28 for immunofluorescence staining of tissuemicroarrays of normal human skin (n=15), benign nevi (n=295), andmalignant melanoma (n=264); representative result shown for each, bluecolor: nuclei, green color: melanocytes, and red color: RNLS. FIG. 17Bis a chart depicting fluorescence intensity quantified using theAQUAnalysis™ software, Yale TMA: normal human skin (n=15), benign nevi(n=295), and malignant melanoma (n=264). FIG. 17C is a chart showingfluorescence intensity quantified using the AQUAnalysis™ software, USBiomax TMA: normal human skin (n=14), benign nevi (n=14), primarymelanoma (n=35), and metastatic melanoma (n=11), * indicates p=0.009 and** indicates p<0.001. FIG. 17D depicts the Kaplan-Meier survival curvefor melanoma-specific death; 119 serial primary melanomas collected from1997 to 2004, tumors stratified into low and high RNLS expression by themedian AQUA score=75,764.45, * indicates p=0.008.

FIG. 18, comprising FIGS. 18A and 18B, is two charts showing that RNLSoverexpression favors cancer cell survival. FIG. 18A is a chartdepicting A375.S2, MeWo, Skme15, and Skme128 cells serum-starved andthen treated with BSA (30 ug/ml) or rRNLS (30 ug/ml), and cell viabilitymeasured 72 hrs later using the WST-1 assay; n=6, * indicates p<0.05 and** indicates p<0.005. FIG. 18B is a chart depicting A375.S2 cellsserum-starved for 24 hrs, then untreated or incubated with 30ug/ml ofeither bovine serum albumin (BSA) or rRNLS for 3 days; total and livecell number were determined using trypan blue and an automated cellcounter; n=6, ** indicates p<0.001.

FIG. 19, comprising FIGS. 19A through 19D, is a series of images andcharts showing that inhibition of RNLS signaling is cytotoxic tomelanoma cells in vitro. FIG. 19A is a chart depicting relative cellviability after transient transfection of melanoma cells A375.S2 andSK-Me1-28 using a RNLS-specific siRNA, or a non-specific control siRNA,where cell viability was assessed 72 hrs later using the WST-1 assay;n=6, * indicates p=0.03 and ** indicates p=0.003. FIG. 19B comprises twocharts depicting relative cell viability: Left panel: Cells were treatedwith indicated antibodies for 72 hrs and cell viability was determinedusing WST-1; m28-RNLS (also known as 1D-28-4), m37-RNLS (also known as1D-37-10): monoclonal antibodies raised against RNLS peptide RP220;Right panel: A375.S2 cells treated with increasing doses of m28-RNLS for72 hrs and cell viability determined with a WST-1 assay; n=6, *indicates p<0.05 and ** indicates p<0.005. FIG. 19C comprisesrepresentative photos of A375.S2, SkMe128, and SkMe15 after 72 hrsincubation with either control rabbit IgG or m28-RNLS. FIG. 19D is achart depicting relative cell viability, comprising amino acid (AA)sequence of RNLS peptide antagonist (RP220A). A375.S2 cells treated withthe indicated concentrations of BSA or RP220A, and cell viabilitymeasured 72 hrs later using the WST-1 assay; n=6, ** indicates p<0.005.

FIG. 20, comprising FIGS. 20A and 20B, comprises a chart and two imagesshowing that inhibition of RNLS signaling blocks melanoma growth invivo. FIG. 20A is a chart showing tumor volume increase in nude athymicmice xenografted with A375.S2 cells, tumor size measured prior totreatment every 3 days with 2 mg/kg of either rabbit IgG as a negativecontrol or with RNLS monoclonal Ab, m28-RNLS; n=14 per group; dailytumor growth rate is computed as change in tumor size from previousmeasurement; * indicates p<0.05. FIG. 20B comprises representativeimages of IHC staining of sections from A375.S2 xenografted tumors (n=14each) treated with m28-RNLS or control rabbit IgG for cell proliferationmarker Ki67; brown color: Ki67 positive cells.

FIG. 21, comprising FIGS. 21A through 21F, is a series of images andcharts showing that inhibition of RNLS signaling blocks RNLS expressionand STAT3 activation and induces apoptosis and cell cycle arrest. FIG.21A comprises a series of images showing xenograft tumors treated witheither rabbit IgG as a negative control or with RNLS monoclonal Ab, andprobed for RNLS, phosphorylated STAT3, and total STAT3 byimmunofluorescence; phospho STAT3=p-Y⁷⁰⁵-STAT3; representative resultshown for each, blue color: nuclei, green color: RNLS, and red color:phospho STAT3 (left panel) or total STAT3 (right panel). FIG. 21B is animage showing xenograft tumors treated with either rabbit IgG as anegative control or with RNLS monoclonal Ab, and tumor cell lysatesprobed for RNLS, phosphorylated STAT3, total STAT3, and p21 by westernblot; p-Y⁷⁰⁵-STAT3: phosphorylation at tyrosine 705; representativestudy. FIG. 21C is a chart depicting quantification of STAT3 proteinexpression in samples shown in FIG. 21B; p-Y⁷⁰⁵-STAT3 signals normalizedto total STAT3, total STAT3 signals normalized to protein loadingmeasurements; n=3, * indicates p<0.05 and ** indicates p<0.005. FIG. 21Dis a chart showing xenograft tumors (n=14 each) treated with eitherrabbit IgG as a negative control or with RNLS monoclonal Ab, and probedfor human and mouse RNLS expression by qPCR, * indicates p<0.05. FIG.21E comprises representative images of IHC staining of sections fromA375.S2 xenografted tumors (n=14 each) treated with m28-RNLS or controlrabbit IgG for TUNEL assay to mark apoptotic cells or cell cycleinhibitor p21; brown color: TUNEL or p21 positive cells, respectively.FIG. 21F is an image showing A375.S2 cells treated with anti-RNLSantibody or control goat IgG; time course of p38 phosphorylation and Baxexpression assessed by western blot; p-p38=phosphorylated p38; Bax=bc1-2like protein 4.

FIG. 22, comprising FIGS. 22A through 22C, is a series of images andcharts showing that RNLS is expressed in CD163+ TAMs in melanoma. FIG.22A comprises images showing: Top panel: Tissue microarray humanmelanoma samples examined by IF for coexpression of RNLS and thepan-macrophage marker CD68; blue color: nuclei, green color: RNLS, andred color: all macrophages; DAPI: nuclear stain, RNLS-CD68: merged RNLSand CD68 stains; Middle panel: Melanoma samples examined by IF forcoexpression of RNLS and the alternatively activated macrophage (M2)marker CD163; blue color: nuclei, green color: RNLS, and red color: M2macrophages; DAPI: nuclear stain, RNLS-CD163: merged RNLS and CD163stains. Significant coexpression of RNLS and CD163 noted; Lower panel:Melanoma samples examined by IF for coexpression of RNLS and theclassically activated macrophage (M1) marker CD86; blue color: nuclei,green color: RNLS, and red color: M1 macrophages; DAPI: nuclear stain,RNLS-CD163: merged RNLS and CD86 stains. No significant coexpression ofRNLS and CD186 noted. FIG. 22B comprises two images showing xenografttumors treated with either rabbit IgG as a negative control or withm28-RNLS, and probed for RNLS and M2 TAMs (CD163+ cells) byimmunofluorescence; representative result shown for each, green color:M2 macrophages, and red color: RNLS. m28-RNLS treatment decreases CD163+TAMs and RNLS expression. FIG. 22C depicts the proposed mechanism ofaction of m28-RNLS- TAM: tumor associated macrophages, CD163:alternatively activated macrophage (M2) marker, CD86: classicallyactivated macrophage (M1) marker, RNLS: renalase, m28-RNLS: antirenalasemonoclonal antibody, t-STAT3: total STAT3, p-STAT3: phosphorylatedSTAT3.

FIG. 23, comprising FIGS. 23A through 23E, is a series of images andcharts showing RNLS expression in some cancers, and association withpoor patient outcome in PDAC. FIG. 23A is a chart showing RNLS mRNAlevel measured by qPCR in cDNA arrays containing 182 human tumor samples(OriGene Technologies) from 15 different tumor types; * indicatesp<0.05, ** indicates p=0.0001. FIG. 23B is a chart showing RNLS mRNAlevel measured by qPCR in normal pancreas (n=6), pancreatic ductaladenocarcinomas (n=11), and pancreatic neuroendocrine tumors (n=23); *indicates p=0.05; ** indicates p=0.00017. FIG. 23C is an image showingRNLS protein expression detected by immunohistochemistry using m28-RNLSin normal human pancreatic tissue (left panel, n=90), ductaladenocarcinoma (Grades 1-4, n=20 each); representative result shown foreach; RNLS protein stains brown. FIG. 23D shows RNLS expression detectedusing anti-RNLS-m28 for immunofluorescence staining of tissue microarrayof normal human pancreatic tissue (left panel, n=90), ductal carcinoma(middle panel, n=90); representative result shown for each, and bluecolor: nuclei, green color: cytokeratin, and red color: RNLS; rightpanel: fluorescence intensity quantified using the AQUAnalysis™software, normal human pancreatic tissue (n=90), ductal carcinoma(n=90), ***indicates p=0.00013. FIG. 23E shows the Kaplan-Meier survivalcurve for survival rates; Biomax cohort of 69 PDACs stratified into low(n=35, RNLS AQUA score <median) and high (n=34, RNLS AQUA score >median)RNLS expression, * indicates p=0.0001.

FIG. 24, comprising FIGS. 24A through 24D, is a series of charts showingthat RNLS overexpression favors cancer cell survival. FIG. 24A showsPDACC lines BxPC3, Panc1 and MiaPaCa2 are serum starved for 48 hrs, thenincubated with 30 μg/ml of either bovine serum albumin (BSA) or rRNLSfor 3 days; total and live cell number determined using trypan blue andan automated cell counter; n=4, ** indicates p<0.0001. FIG. 24B is achart depicting cell viability relative to control: MiaPaCa2 cell serumstarved and then treated with BSA (30 μg/ml) or rRNLS (30 μg/ml), withand without pretreatment with MEK1 inhibitor U0126, and cell viabilitymeasured 72 hrs later using the WST-1 assay; n=6, * indicates p<0.005.FIG. 24C comprises an image and a chart showing that siRNA mediatedinhibition of PMCA4b expression blocks RNLS mediated MAPK signaling;Left and middle panels: MiaPaCa2 cells transfected with eithernon-targeting or PMCA4b siRNA, maintained in serum free medium for 3days and treated with either 25 μg of BSA or 25 of RNLS peptide RP-220for the indicated time; RP-220 mediated ERK and STAT3 activationassessed by western blot and representative immunoblots are shown;p-ERK=phosphorylated ERK, p-Y⁷⁰⁵-STAT3=phosphorylated STAT3,p-S727-STAT3=phosphorylated STAT3, BSA=bovine serum albumin, RP-220=RNLSpeptide agonist; Right panel: quantification of phosphorylated ERK(p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase(GAPDH) loading control; n=3, * =P<0.05.

FIG. 24D shows is a graph showing fluorescence activated cell sorting(FACS) analysis of MiaPaCa2 cells treated with BSA (30 μg/ml) or rRNLS(30 μg/ml), n=3.

FIG. 25, comprising FIGS. 25A through 25E, is a series of charts andimages showing that inhibition of RNLS signaling is cytotoxic to cancercells in vitro and in vivo. FIG. 25A is a chart showing relative cellviability following transient transfection of Panc1 cells using aRNLS-specific siRNA, or a non-specific control siRNA, and cell viabilityassayed 96h later using the WST-1 reagent; n=6, ** indicates p<0.001.FIG. 25B is a chart showing relative cell viability when cells weretreated with indicated antibodies for 72 hrs and cell viabilitydetermined using WST-1; m28-RNLS and m37-RNLS: monoclonal antibodiesraised against RNLS peptide RP220, ab31291: Abcam polyclonal antibodyraised against a partial sequence of RP-220; n=6, * indicates p<0.005.FIG. 25C shows representative photos of MiaPaCa2 cells after 3 daysincubation with m28-RNLS, n=10. FIG. 25D is a chart showing tumor volumeincrease after athymic nude mice received subcutaneous injection ofPanc1 cells transduced with RNLS shRNA (sh-RNLS) or control(sh-Control); tumor volume measured every 23 days for up to 30 days, n=6each; * indicates p<0.05. FIG. 25E is chart showing tumor volumeincrease after nude mice were xenografted with BxPC3; tumor volumemeasured prior to treatment every 3-4 days with 2 mg/kg of either rabbitIgG as a negative control or with m28-RNLS, n=10, * indicates p<0.05.

FIG. 26, comprising FIGS. 26A through 26E, is a series of images andcharts showing that inhibition of RNLS signaling induces apoptosis andcell cycle arrest. FIG. 26A shows representative images of TUNELstaining of sections from BxPC3 xenografted tumors (n=14 each) treatedwith anti-m28-RNLS or control rabbit IgG; arrow: TUNEL positive cells.FIG. 26B is a chart depicting FACS analysis of Panc1 cells in culturetreated with either m28-RNLS (30 μg/ml) or 100 μM etoposide (positivecontrol) for 4 days; n=3, * indicates p<0.05. FIG. 26C is an imageshowing Panc1 cells treated with polyclonal ab31291 or with goat IgG asa negative control, and cells lysates probed for p38 and Bax activationby western blot. FIG. 26D shows representative images of IHC staining ofsections from BxPC3 xenografted tumors (n=14 each) treated withanti-m28-RNLS or control rabbit IgG for cell proliferation marker ki67,and cell cycle inhibitor p21. FIG. 26E is a chart showing the effect ofm28-RNLS on cell cycle of Panc1 cells determined by FACS analysis; greencurve: no treatment, purple curve: rabbit IgG, red curve: m28-RNLS 30μg/ml.

FIG. 27, comprising FIGS. 27A through 27E, is a series of images andcharts showing the interaction between RNLS and STAT3, and themechanistic model of inhibition by m28-RNLS. FIG. 27A is an imageshowing activation of STAT3 by RNLS in Panc1 cells; Panc1 cells inculture treated with either BSA or RNLS, and STAT3 phosphorylationassessed by western blot; p-Ser⁷²⁷-STAT3: phosphorylation at serine 727,p-Y⁷⁰⁵-STAT3: phosphorylation at tyrosine 705; representative study.FIG. 27B is a chart depicting the quantification of STAT3phosphorylation with RNLS; signals normalized to total STAT3; n=3,*=P<0.05. FIG. 27C is an image showing that m28-RNLS inhibits STAT3phosphorylation; Panc1 cells in culture treated with either rabbit IgGor anti-RNLS monoclonal m28-RNLS for up to 4 days, and STAT3phosphorylation assessed by western blot; p-Ser⁷²⁷-STAT3:phosphorylation at serine 727, p-Y⁷⁰⁵-STAT3: phosphorylation at tyrosine705; GAPDH loading control; representative study. FIG. 27D is a chartshowing the quantification of STAT3 phosphorylation with m28-RNLS;signals normalized to GAPDH loading control; n=3, *=P<0.05. FIG. 27Edepicts the proposed mechanistic model for antitumor activity ofm28-RNLS.

FIG. 28 comprises images depicting that RNLS expression was present inPDAC grade 1-4 and was predominantly localized to cancer cells.

FIG. 29 comprises images showing RNLS expression in neuroendocrine tumorof the pancreas, and showing that RNLS was expressed in cells throughoutthe tumor.

FIG. 30 is a chart depicting the relative RNLS mRNA levels normalized toβ-actin, showing that RNLS gene expression was greater in pancreaticductal adenocarcinoma cell (PDACC) lines with KRAS mutations (MiaPaCa2and Panc1) than those with wild type KRAS, such as BxPC3.

FIG. 31 is a chart depicting the relative RNLS mRNA levels normalizedwith β-actin, showing the effect of decreasing RNLS expression on cellviability in vitro, evaluated by RNLS knockdown by siRNA; this treatmentmarkedly reduced the viability of the PDACC lines Panc1 and MiaPaCa2.

FIG. 32 is a chart showing that inhibition of RNLS expression byRNLS-targeting shRNA resulted in a marked reduction in the expression ofits receptor PMCA4b, suggesting RNLS and PMCA4b expression areco-regulated.

FIG. 33 is a series of charts depicting FACS analysis of Panc1 cells inculture, which confirmed m28-RNLS caused apoptosis.

FIG. 34 comprises an image and a chart, showing that a positiveRNLS-STAT3 feedback loop is suggested by the observation that in HK-2cells treated with RNLS, STAT3 phosphorylation at serine 727(p-Ser⁷²⁷-STAT3) and tyrosine 705 (p-Y⁷⁰⁵-STAT3) increases 2 and 4 foldrespectively, but STAT1 is unaffected.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the inhibition of renalase using an inhibitorof renalase. In various embodiments, the invention is directed tocompositions and methods for treating a renalase-associated pathology orrenalase-associated condition in an individual by administering to asubject in need thereof an inhibitor of renalase. In variousembodiments, the diseases and disorders diagnosable, preventable andtreatable using the compositions and methods of the invention includeacute renal failure (i.e., acute tubular necrosis, or ATN, an ischemiccondition in the kidney), cardiovascular disease, and cancer.

In one embodiment, the invention broadly relates to the treatment,prevention, and diagnosis of cancer. In one embodiment, the presentinvention is directed to methods and compositions for diagnosis,treatment, inhibition, prevention, or reduction of cancer. In oneembodiment, the invention provides compositions and methods formodulating one or more of the level, production, and activity ofrenalase. In the context of cancer and related diseases and disorders,the invention provides compositions and methods for decreasing one ormore of the level, production, and activity of renalase. Some aspects ofthe invention provide methods and compositions for the treatment,prevention, diagnosis or prognosis of cancer metastasis.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

Generally, the nomenclature used herein and the laboratory procedures incell culture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al.,2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used inanalytical chemistry and organic syntheses described below are thosewell-known and commonly employed in the art. Standard techniques ormodifications thereof are used for chemical syntheses and chemicalanalyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected/homeostatic) respective characteristic.Characteristics which are normal or expected for one cell, tissue type,or subject, might be abnormal for a different cell or tissue type.

The term “analog” as used herein generally refers to compounds that aregenerally structurally similar to the compound of which they are ananalog, or “parent” compound. Generally analogs will retain certaincharacteristics of the parent compound, e.g., a biological orpharmacological activity. An analog may lack other, less desirablecharacteristics, e.g., antigenicity, proteolytic instability, toxicity,and the like. An analog includes compounds in which a particularbiological activity of the parent is reduced, while one or more distinctbiological activities of the parent are unaffected in the “analog.” Asapplied to polypeptides, the term “analog” may have varying ranges ofamino acid sequence identity to the parent compound, for example atleast about 70%, more preferably at least about 80%-85% or about86%-89%, and still more preferably at least about 90%, about 92%, about94%, about 96%, about 98% or about 99% of the amino acids in a givenamino acid sequence the parent or a selected portion or domain of theparent. As applied to polypeptides, the term “analog” generally refersto polypeptides which are comprised of a segment of about at least 3amino acids that has substantial identity to at least a portion of abinding domain fusion protein. Analogs typically are at least 5 aminoacids long, at least 20 amino acids long or longer, at least 50 aminoacids long or longer, at least 100 amino acids long or longer, at least150 amino acids long or longer, at least 200 amino acids long or longer,and more typically at least 250 amino acids long or longer. Some analogsmay lack substantial biological activity but may still be employed forvarious uses, such as for raising antibodies to predetermined epitopes,as an immunological reagent to detect and/or purify reactive antibodiesby affinity chromatography, or as a competitive or noncompetitiveagonist, antagonist, or partial agonist of a binding domain fusionprotein function.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope of abinding partner molecule. Antibodies can be intact immunoglobulinsderived from natural sources, or from recombinant sources and can beimmunoreactive portions of intact immunoglobulins. The antibodies in thepresent invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, intracellularantibodies (“intrabodies”), Fv, Fab, Fab′, F(ab)2 and F(ab′)2, as wellas single chain antibodies (scFv), heavy chain antibodies, such ascamelid antibodies, and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to at least one portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,sdAb (either V_(L) or V_(H)), camelid V_(HH) domains, scFv antibodies,and multi-specific antibodies formed from antibody fragments. The term“scFv” refers to a fusion protein comprising at least one antibodyfragment comprising a variable region of a light chain and at least oneantibody fragment comprising a variable region of a heavy chain, whereinthe light and heavy chain variable regions are contiguously linked via ashort flexible polypeptide linker, and capable of being expressed as asingle chain polypeptide, and wherein the scFv retains the specificityof the intact antibody from which it was derived. Unless specified, asused herein an scFv may have the V_(L) and V_(H) variable regions ineither order, e.g., with respect to the N-terminal and C-terminal endsof the polypeptide, the scFv may comprise V_(L)-linker-V_(H) or maycomprise V_(H)-linker-V_(L).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations, and which normally determines theclass to which the antibody belongs.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations. Kappa (κ) and lambda (λ) light chainsrefer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., 1989, Queen etal., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al.,Bio/Technology, 9:421). A suitable human acceptor antibody may be oneselected from a conventional database, e.g., the KABAT database, LosAlamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanized antibodies (see for example EP-A-0239400 andEP-A-054951).

The term “donor antibody” refers to an antibody (monoclonal, and/orrecombinant) which contributes the amino acid sequences of its variableregions, CDRs, or other functional fragments or analogs thereof to afirst immunoglobulin partner, so as to provide the alteredimmunoglobulin coding region and resulting expressed altered antibodywith the binding specificity and neutralizing activity characteristic ofthe donor antibody.

The term “acceptor antibody” refers to an antibody (monoclonal and/orrecombinant) heterologous to the donor antibody, which contributes all(or any portion, but in some embodiments all) of the amino acidsequences encoding its heavy and/or light chain framework regions and/orits heavy and/or light chain constant regions to the firstimmunoglobulin partner. In certain embodiments a human antibody is theacceptor antibody.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883.

The term “framework” or “framework sequence” refers to the remainingsequences of a variable region minus the CDRs. Because the exactdefinition of a CDR sequence may be determined by different systems, themeaning of a framework sequence is subject to correspondingly differentinterpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain andCDR-H1, -H2, and -H3 of heavy chain) also divide the framework regionson the light chain and the heavy chain into four sub-regions (FR1, FR2,FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 andFR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. An FR represents one of the four sub-regions, andFRs represents two or more of the four sub-regions constituting aframework region.

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific bindingpartner molecule, but does not substantially recognize or bind othermolecules in a sample. For example, an antibody that specifically bindsto a binding partner molecule from one species may also bind to thatbinding partner molecule from one or more species. But, suchcross-species reactivity does not itself alter the classification of anantibody as specific. In another example, an antibody that specificallybinds to binding partner molecule may also bind to different allelicforms of the binding partner molecule. However, such cross reactivitydoes not itself alter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specificallybinding”, can be used in reference to the interaction of an antibody, aprotein, or a peptide with a second binding partner molecule, to meanthat the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the bindingpartner molecule; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody. In some instances, the terms “specific binding” and“specifically binding” refers to selective binding, wherein the antibodyrecognizes a sequence or conformational epitope important for theenhanced affinity of binding to the binding partner molecule.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a renalase when a binding protein specificallybinds the renalase. Preferably a neutralizing binding protein is aneutralizing antibody, the binding of which to renalase results ininhibition of a biological activity of renalase. Preferably theneutralizing binding protein binds renalase and reduces a biologicallyactivity of renalase by at least about 20%, 40%, 60, 80%, 85% or more.In some embodiments, the renalase is human renalase.

The term “epitope” has its ordinary meaning of a site on binding partnermolecule recognized by an antibody or a binding portion thereof or otherbinding molecule, such as, for example, an scFv. Epitopes may bemolecules or segments of amino acids, including segments that representa small portion of a whole protein or polypeptide. Epitopes may beconformational (i.e., discontinuous). That is, they may be formed fromamino acids encoded by noncontiguous parts of a primary sequence thathave been juxtaposed by protein folding.

The phrase “biological sample” as used herein, is intended to includeany sample comprising a cell, a tissue, or a bodily fluid in whichexpression of a nucleic acid or polypeptide can be detected. Examples ofsuch biological samples include but are not limited to blood, lymph,bone marrow, biopsies and smears. Samples that are liquid in nature arereferred to herein as “bodily fluids.” Biological samples may beobtained from a patient by a variety of techniques including, forexample, by scraping or swabbing an area or by using a needle to obtainbodily fluids. Methods for collecting various body samples are wellknown in the art.

The term “cancer” as used herein is defined as disease characterized bythe abnormal growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers include but are not limited to, breastcancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer(e.g., melanoma), pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma andthe like.

As used herein, “conjugated” refers to covalent attachment of onemolecule to a second molecule.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of a mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anti-codonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding region may thusinclude nucleotide residues comprising codons for amino acid residueswhich are not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

“Complementary” as used herein to refer to a nucleic acid, refers to thebroad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. Itis known that an adenine residue of a first nucleic acid region iscapable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an antiparallel fashion, at least about 50%,and preferably at least about 75%, at least about 90%, or at least about95% of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. More preferably,all nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

As used herein, the term “derivative” includes a chemical modificationof a polypeptide, polynucleotide, or other molecule. In the context ofthis invention, a “derivative polypeptide,” for example, one modified byglycosylation, pegylation, or any similar process, retains bindingactivity. For example, the term “derivative” of binding domain includesbinding domain fusion proteins, variants, or fragments that have beenchemically modified, as, for example, by addition of one or morepolyethylene glycol molecules, sugars, phosphates, and/or other suchmolecules, where the molecule or molecules are not naturally attached towild-type binding domain fusion proteins. A “derivative” of apolypeptide further includes those polypeptides that are “derived” froma reference polypeptide by having, for example, amino acidsubstitutions, deletions, or insertions relative to a referencepolypeptide. Thus, a polypeptide may be “derived” from a wild-typepolypeptide or from any other polypeptide. As used herein, a compound,including polypeptides, may also be “derived” from a particular source,for example from a particular organism, tissue type, or from aparticular polypeptide, nucleic acid, or other compound that is presentin a particular organism or a particular tissue type.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting there from. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a sign orsymptom of the disease or disorder, the frequency with which such a signor symptom is experienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.

The term “high affinity” for binding domain polypeptides describedherein refers to a dissociation constant (Kd) of at least about 10⁻⁶M,preferably at least about 10⁻⁷M, more preferably at least about 10⁻⁸M orstronger, more preferably at least about 10⁻⁹M or stronger, morepreferably at least about 10⁻¹° M or stronger, for example, up to 10⁻¹²M or stronger. However, “high affinity” binding can vary for otherbinding domain polypeptides.

The term “inhibit,” as used herein, means to suppress or block anactivity or function, for example, about ten percent relative to acontrol value. Preferably, the activity is suppressed or blocked by 50%compared to a control value, more preferably by 75%, and even morepreferably by 95%. “Inhibit,” as used herein, also means to reduce thelevel of a molecule, a reaction, an interaction, a gene, an mRNA, and/ora protein's expression, stability, function or activity by a measurableamount or to prevent entirely. Inhibitors are compounds that, e.g., bindto, partially or totally block activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

The terms “modulator” and “modulation” of a molecule of interest, asused herein in its various forms, is intended to encompass antagonism,agonism, partial antagonism and/or partial agonism of an activityassociated the protease of interest. In various embodiments,“modulators” may inhibit or stimulate protease expression or activity.Such modulators include small molecules agonists and antagonists of aprotease molecule, antisense molecules, ribozymes, triplex molecules,and RNAi polynucleotides, and others.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in its normal context in aliving animal is not “isolated,” but the same nucleic acid or peptidepartially or completely separated from the coexisting materials of itsnatural context is “isolated.” An isolated nucleic acid or protein canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine. The term “polynucleotide” asused herein is defined as a chain of nucleotides. Furthermore, nucleicacids are polymers of nucleotides. Thus, nucleic acids andpolynucleotides as used herein are interchangeable. One skilled in theart has the general knowledge that nucleic acids are polynucleotides,which can be hydrolyzed into the monomeric “nucleotides.” The monomericnucleotides can be hydrolyzed into nucleosides. As used hereinpolynucleotides include, but are not limited to, all nucleic acidsequences which are obtained by any means available in the art,including, without limitation, recombinant means, i.e., the cloning ofnucleic acid sequences from a recombinant library or a cell genome,using ordinary cloning technology and PCR, and the like, and bysynthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the activity of the polypeptide, i.e.,substitution of amino acids with other amino acids having similarproperties. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are generally understood to representconservative substitutions for one another: (1) Alanine (A), Serine (S),Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine(N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W) (see also, Creighton, 1984, Proteins, W.H.Freeman and Company). In addition to the above-defined conservativesubstitutions, other modifications of amino acid residues can alsoresult in “conservatively modified variants.” For example, one mayregard all charged amino acids as substitutions for each other whetherthey are positive or negative. In addition, conservatively modifiedvariants can also result from individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids, for example, often less than 5%, in anencoded sequence. Further, a conservatively modified variant can be madefrom a recombinant polypeptide by substituting a codon for an amino acidemployed by the native or wild-type gene with a different codon for thesame amino acid.

The term “RNA” as used herein is defined as ribonucleic acid. The term“recombinant DNA” as used herein is defined as DNA produced by joiningpieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

By “pharmaceutically acceptable” it is meant, for example, a carrier,diluent or excipient that is compatible with the other ingredients ofthe formulation and generally safe for administration to a recipientthereof. As used herein, “pharmaceutically acceptable carrier” includesany material, which when combined with the conjugate retains theconjugates' activity and is non-reactive with the subject's immunesystems. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid or salts thereof, magnesium or calcium stearate,talc, vegetable fats or oils, gums, glycols, or other known excipients.Such carriers may also include flavor and color additives or otheringredients. Compositions comprising such carriers are formulated bywell-known conventional methods.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, preferably a mammal,and most preferably a human, having a complement system, including ahuman in need of therapy for, or susceptible to, a condition or itssequelae. Thus, the individual may include, for example, dogs, cats,pigs, cows, sheep, goats, horses, rats, monkeys, and mice and humans.

The phrase “percent (%) identity” refers to the percentage of sequencesimilarity found in a comparison of two or more amino acid sequences.Percent identity can be determined electronically using any suitablesoftware. Likewise, “similarity” between two polypeptides (or one ormore portions of either or both of them) is determined by comparing theamino acid sequence of one polypeptide to the amino acid sequence of asecond polypeptide. Any suitable algorithm useful for such comparisonscan be adapted for application in the context of the invention.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

“Therapeutically effective amount” is an amount of a compound of theinvention, that when administered to a patient, ameliorates a symptom ofthe disease. The amount of a compound of the invention which constitutesa “therapeutically effective amount” will vary depending on thecompound, the disease state and its severity, the age of the patient tobe treated, and the like. The therapeutically effective amount can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, acomposition of the present invention, for example, a subject afflicted adisease or disorder, or a subject who ultimately may acquire such adisease or disorder, in order to prevent, cure, delay, reduce theseverity of, or ameliorate one or more symptoms of the disorder orrecurring disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialbiological properties of the reference molecule. Changes in the sequenceof a nucleic acid variant may not alter the amino acid sequence of apeptide encoded by the reference nucleic acid, or may result in aminoacid substitutions, additions, deletions, fusions and truncations.Changes in the sequence of peptide variants are typically limited orconservative, so that the sequences of the reference peptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference peptide can differ in amino acid sequence by oneor more substitutions, additions, deletions in any combination. Avariant of a nucleic acid or peptide can be a naturally occurring suchas an allelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

This invention relates to the inhibition of renalase using an inhibitorof renalase. In various embodiments, the invention is directed tocompositions and methods for treating a renalase-associated disease ordisorder in an individual by administering to a subject in need thereofan inhibitor of renalase. In some embodiments, the renalase inhibitor isa renalase binding molecule. In some embodiments, the renalase bindingmolecule is an antibody. In various embodiments, the diseases anddisorders diagnosable, preventable and treatable using the compositionsand methods of the invention include acute renal failure (i.e., acutetubular necrosis, or ATN, an ischemic condition in the kidney),cardiovascular disease, and cancer.

In one embodiment, the invention broadly relates to the treatment,prevention, and diagnosis of cancer. In one embodiment, the presentinvention is directed to methods and compositions for diagnosis,staging, treatment, inhibition, prevention, or reduction of cancer. Inone embodiment, the invention provides compositions and methods formodulating one or more of the level, production, and activity ofrenalase. In the context of cancer and related diseases and disorders,the invention provides compositions and methods for decreasing one ormore of the level, production, and activity of renalase. Some aspects ofthe invention provide methods and compositions for the treatment,prevention, diagnosis or prognosis of cancer metastasis.

Therapeutic Inhibitor Compositions and Methods of Use

In various embodiments, the present invention includes renalaseinhibitor compositions and methods of treating or preventing a diseaseor disorder where a diminished level or activity of renalase is desired.One non-limiting example of a disease or disorder where a diminishedlevel or activity of renalase is desired which can be treated orprevented with the compositions and methods of the invention includescancer. In various embodiments, the renalase inhibitor compositions andmethods of treatment or prevention of the invention diminish the amountof renalase polypeptide, the amount of renalase peptide fragment, theamount of renalase mRNA, the amount of renalase enzymatic activity, theamount of renalase substrate binding activity, the amount of renalasereceptor binding activity, or a combination thereof.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that a decrease in the level of renalaseencompasses the decrease in renalase expression, includingtranscription, translation, or both, and also encompasses promoting thedegradation of renalase, including at the RNA level (e.g., RNAi, shRNA,etc.) and at the protein level (e.g., Ubiquitination, etc.) The skilledartisan will also appreciate, once armed with the teachings of thepresent invention, that a decrease in the level of renalase includes adecrease in a renalase activity (e.g., enzymatic activity, substratebinding activity, receptor binding activity, etc.). Thus, decreasing thelevel or activity of renalase includes, but is not limited to,decreasing transcription, translation, or both, of a nucleic acidencoding renalase; and it also includes decreasing any activity of arenalase polypeptide, or peptide fragment thereof, as well. The renalaseinhibitor compositions and methods of the invention can selectivelyinhibit renalase, or can inhibit both renalase and another molecule.

Inhibition of renalase can be assessed using a wide variety of methods,including those disclosed herein, as well as methods known in the art orto be developed in the future. That is, the routineer would appreciate,based upon the disclosure provided herein, that decreasing the level oractivity of renalase can be readily assessed using methods that assessthe level of a nucleic acid encoding renalase (e.g., mRNA), the level ofa renalase polypeptide, or peptide fragment thereof, present in abiological sample, the level of renalase activity (e.g., enzymaticactivity, substrate binding activity, receptor binding activity, etc.),or combinations thereof.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in treating or preventing in asubject in need thereof, whether or not the subject is also beingtreated with other medication or therapy. Further, the skilled artisanwould further appreciate, based upon the teachings provided herein, thatthe disease or disorders treatable by the compositions and methodsdescribed herein encompass any disease or disorder where renalase playsa role and where diminished renalase level or activity will promote apositive therapeutic outcome. In various embodiments, the disease ordisorder treatable or preventable using the compounds and methods of theinvention include acute renal failure (i.e., acute tubular necrosis, orATN, an ischemic condition in the kidney), a cardiovascular disease ordisorder (e.g., hypertension, pulmonary hypertension, systolichypertension, diabetic hypertension, asymptomatic left ventriculardysfunction, chronic congestive heart failure, myocardial infarction,cardiac rhythm disturbance, atherosclerosis, etc.), cancer, heartdisease or disorder, a kidney disease or disorder, a gastrointestinaldisease or disorder, a liver disease or disorder, a lung disease ordisorder, a pancreas disease or disorder (e.g., pancreatitis), mentaldisease or disorder (e.g., depression, anxiety, etc.), or a neurologicaldisease or disorder.

In another embodiment, the renalase inhibitor of the invention can beadministered to a patient who is being treated with exogenous renalase,recombinant renalase, renalase fragment, and/or renalase activator, inorder to control, titrate, diminish, or stabilize the level or activityof endogenous and/or exogenous renalase in the patient.

The renalase inhibitor compositions and methods of the invention thatdecrease the level or activity (e.g., enzymatic activity, substratebinding activity, receptor binding activity, etc.) of renalase, or arenalase fragment, include, but should not be construed as being limitedto, a chemical compound, a protein, a peptide, a peptidomemetic, anantibody, an antibody fragment, an antibody mimetic, a ribozyme, a smallmolecule chemical compound, an short hairpin RNA, RNAi, an antisensenucleic acid molecule (e.g., siRNA, miRNA, etc.), a nucleic acidencoding an antisense nucleic acid molecule, a nucleic acid sequenceencoding a protein, a renalase receptor, a renalase receptor fragment,or combinations thereof. In some embodiments, the inhibitor is anallosteric inhibitor. One of skill in the art would readily appreciate,based on the disclosure provided herein, that a renalase inhibitorcomposition encompasses any chemical compound that decreases the levelor activity of renalase, or a fragment thereof. Additionally, a renalaseinhibitor composition encompasses a chemically modified compound, andderivatives, as is well known to one of skill in the chemical arts.

The renalase inhibitor compositions and methods of the invention thatdecrease the level or activity (e.g., enzymatic activity, substratebinding activity, receptor binding activity, etc.) of renalase, or arenalase fragment, include antibodies, and fragments thereof. Theantibodies of the invention include a variety of forms of antibodiesincluding, for example, polyclonal antibodies, monoclonal antibodies,intracellular antibodies (“intrabodies”), Fv, Fab and F(ab)2, singlechain antibodies (scFv), heavy chain antibodies (such as camelidantibodies), synthetic antibodies, chimeric antibodies, and a humanizedantibodies. In one embodiment, the antibody of the invention is anantibody that specifically binds to renalase. In some embodiments, theantibodies of the invention are bispecific antibodies, where the firstspecificity is to renalase and the second specificity is to a targetingmolecule on a cell or tissue to guide the bispecific antibody to ananatomic location where the targeting molecule is present and where therenalase binding is desired. In some embodiments, the antibodies of theinvention are bispecific antibodies, where the first specificity is torenalase and the second specificity is to a second binding partnermolecule (i.e., payload) that is carried by the antibodies secondspecificity and deployed to an anatomic location where renalase bindingis desired.

In some embodiments, the administration to the subject of the renalaseinhibitor (e.g., renalase binding molecule) of the invention for thetreatment of cancer, serves to initiate and/or supplement an immuneresponse by the subject's immune system against the cancer. Thesubject's immune response against the cancer can be any host defense orresponse, including an innate immune response, a humoral immuneresponse, a cell-mediated immune response, or a combination thereof.

Further, one of skill in the art, when equipped with this disclosure andthe methods exemplified herein, would appreciate that a renalaseinhibitor composition includes such inhibitors as discovered in thefuture, as can be identified by well-known criteria in the art ofpharmacology, such as the physiological results of inhibition ofrenalase as described in detail herein and/or as known in the art.Therefore, the present invention is not limited in any way to anyparticular renalase inhibitor composition as exemplified or disclosedherein; rather, the invention encompasses those inhibitor compositionsthat would be understood by the routineer to be useful as are known inthe art and as are discovered in the future.

Further methods of identifying and producing renalase inhibitorcompositions are well known to those of ordinary skill in the art,including, but not limited, obtaining an inhibitor from a naturallyoccurring source (e.g., Streptomyces sp., Pseudomonas sp., Stylotellaaurantium, etc.). Alternatively, a renalase inhibitor can be synthesizedchemically. Further, the routineer would appreciate, based upon theteachings provided herein, that a renalase inhibitor composition can beobtained from a recombinant organism. Compositions and methods forchemically synthesizing renalase inhibitors and for obtaining them fromnatural sources are well known in the art and are described in the art.

One of skill in the art will appreciate that an inhibitor can beadministered as a chemical compound, a protein, a peptide, apeptidomemetic, an antibody, an antibody fragment, an antibody mimetic,a ribozyme, a small molecule chemical compound, an short hairpin RNA,RNAi, an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), anucleic acid encoding an antisense nucleic acid molecule, a nucleic acidsequence encoding a protein, a renalase receptor, a renalase receptorfragment, or combinations thereof. Numerous vectors and othercompositions and methods are well known for administering a protein or anucleic acid construct encoding a protein to cells or tissues.Therefore, the invention includes a method of administering a protein ora nucleic acid encoding a protein that is an inhibitor of renalase.(Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself increases the level or activity ofrenalase can serve in the compositions and methods of the presentinvention to decrease the level or activity of renalase.

Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to some portion of an RNA molecule. When present in acell, antisense oligonucleotides hybridize to an existing RNA moleculeand inhibit translation into a gene product. Inhibiting the expressionof a gene using an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of an antisenseoligonucleotide to diminish the amount of renalase, or to diminish theamount of a molecule that causes an increase in the amount or activityof renalase, thereby decreasing the amount or activity of renalase.

Contemplated in the present invention are antisense oligonucleotidesthat are synthesized and provided to the cell by way of methods wellknown to those of ordinary skill in the art. As an example, an antisenseoligonucleotide can be synthesized to be between about 10 and about 100,more preferably between about 15 and about 50 nucleotides long. Thesynthesis of nucleic acid molecules is well known in the art, as is thesynthesis of modified antisense oligonucleotides to improve biologicalactivity in comparison to unmodified antisense oligonucleotides (Tullis,1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing renalase, or of a geneexpressing a protein that increases the level or activity of renalase,can be accomplished through the use of a ribozyme. Using ribozymes forinhibiting gene expression is well known to those of skill in the art(see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al.,1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).Ribozymes are catalytic RNA molecules with the ability to cleave othersingle-stranded RNA molecules. Ribozymes are known to be sequencespecific, and can therefore be modified to recognize a specificnucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowingthe selective cleavage of specific mRNA molecules. Given the nucleotidesequence of the molecule, one of ordinary skill in the art couldsynthesize an antisense oligonucleotide or ribozyme without undueexperimentation, provided with the disclosure and referencesincorporated herein.

Alternatively, inhibition of a gene expressing renalase, or of a geneexpressing a protein that increases the level or activity of renalase,can be accomplished through the use of a short hairpin RNA or antisenseRNA, including siRNA, miRNA, and RNAi. Given the nucleotide sequence ofthe molecule, one of ordinary skill in the art could synthesize such anshort hairpin RNA or antisense RNA without undue experimentation,provided with the disclosure and references incorporated herein.

One of skill in the art will appreciate that inhibitors of renalase, ora renalase fragment, can be administered acutely (e.g., over a shortperiod of time, such as a day, a week or a month) or chronically (e.g.,over a long period of time, such as several months or a year or more).One of skill in the art will appreciate that inhibitors of renalase canbe administered singly or in any combination with other agents. Further,renalase inhibitors can be administered singly or in any combination ina temporal sense, in that they may be administered concurrently, orbefore, and/or after each other. One of ordinary skill in the art willappreciate, based on the disclosure provided herein, that renalaseinhibitor compositions can be used to treat or prevent a disease ordisorder in a subject in need thereof, and that an inhibitor compositioncan be used alone or in any combination with another inhibitor to effecta therapeutic result.

In various embodiments, any of the inhibitors of renalase, or renalasefragment, of the invention described herein can be administered alone orin combination with other inhibitors of other molecules associated withcancer.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder, such ascancer, that is already established. Particularly, the disease ordisorder need not have manifested to the point of detriment to thesubject; indeed, the disease or disorder need not be detected in asubject before treatment is administered. That is, significant diseaseor disorder does not have to occur before the present invention mayprovide benefit. Therefore, the present invention includes a method forpreventing a disease or disorder in a subject, in that a renalaseinhibitor composition, as discussed previously elsewhere herein, can beadministered to a subject prior to the onset of the disease or disorder,thereby preventing the disease or disorder from developing. Thepreventive methods described herein also include the treatment of asubject that is in remission for the prevention of a recurrence of adisease or disorder.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of a disease or disorder encompassesadministering to a subject a renalase inhibitor composition as apreventative measure against the disease or disorder, including cancer.As more fully discussed elsewhere herein, methods of decreasing thelevel or activity of renalase encompass a wide plethora of techniquesfor decreasing not only renalase activity, but also for decreasingexpression of a nucleic acid encoding renalase, including either adecrease in transcription, a decrease in translation, or both.

Additionally, as disclosed elsewhere herein, one skilled in the artwould understand, once armed with the teaching provided herein, that thepresent invention encompasses a method of preventing a wide variety ofdiseases, disorders and pathologies where a decrease in expressionand/or activity of renalase mediates, treats or prevents the disease,disorder or pathology. Methods for assessing whether a disease relatesto the levels or activity of renalase are known in the art. Further, theinvention encompasses treatment or prevention of such diseasesdiscovered in the future.

The invention encompasses administration of an inhibitor of renalase topractice the methods of the invention; the skilled artisan wouldunderstand, based on the disclosure provided herein, how to formulateand administer the appropriate renalase inhibitor to a subject. However,the present invention is not limited to any particular method ofadministration or treatment regimen.

The invention provides compositions that bind to renalase. In oneembodiment, the renalase binding agent inhibits renalase levels oractivity. Thus, in diseases and conditions where a reduction of renalaseactivity would be beneficial, such inhibitory renalase binding agentscan potentially act as therapeutics.

In some instances, in addition to its potential therapeutic role,renalase can be used as a diagnostic marker for diseases or disordersincluding, but not limited, to acute renal failure (i.e., acute tubularnecrosis, or ATN, an ischemic condition in the kidney), cardiovasculardisease, and cancer. Patients without a properly functioning kidneypossess lower levels of renalase. Accordingly, also included in theinvention are methods of diagnosing susceptibility to cardiovascular,heart, kidney, gastrointestinal, liver, lung, pancreas and mental andneurological related conditions, disorders and diseases, includingcancer, based on the detection and/or quantitation of renalase using therenalase binding agents of the present invention. For example,cardiovascular conditions, disorders and diseases such as hypertension,asymptomatic left ventricular dysfunction, chronic congestive heartfailure, myocardial infarction, cardiac rhythm disturbance, andatherosclerosis; mental conditions, disorders and diseases such asdepression and anxiety; and heart conditions, disorders and diseases,such as pulmonary hypertension, can all be diagnosed, evaluated andmonitored by determining renalase levels, such as renalase proteinlevels. For example, reduced levels of the renalase protein would be adiagnostic marker for a disorder associated with an increasedsympathetic output. The compositions and methods of the presentinvention can be used to treat, prevent, reduce or amelioratehypertension, including systolic hypertension, isolated systolichypertension and diabetic hypertension. Moreover, the same benefit isanticipated for the more rare hypertensive disorder, pulmonaryhypertension, as well as pancreatitis. Pulmonary hypertension is a rareblood vessel disorder of the lung in which the pressure in the pulmonaryartery (the blood vessel that leads from the heart to the lungs) risesabove normal levels and may become life threatening. The similarity indevelopment of elevated blood pressure in the pulmonary bed with theincrease in systemic blood pressure in diabetic hypertension and inisolated systolic hypertension suggests similar mechanisms are involved.

The renalase inhibitor compositions of the invention that decrease thelevel or activity (e.g., enzymatic activity, substrate binding activity,receptor binding activity, etc.) of renalase include, but should not beconstrued as being limited to, a chemical compound, a protein, apeptide, a peptidomemetic, an antibody, an antibody fragment, anantibody mimetic, a ribozyme, a small molecule chemical compound, anshort hairpin RNA, RNAi, an antisense nucleic acid molecule (e.g.,siRNA, miRNA, etc.), a nucleic acid encoding an antisense nucleic acidmolecule, a nucleic acid sequence encoding a protein, a renalasereceptor, a renalase receptor fragment, or combinations thereof. In someembodiments, the inhibitor is an allosteric inhibitor. One of skill inthe art would readily appreciate, based on the disclosure providedherein, that a renalase inhibitor composition encompasses a chemicalcompound that decreases the level or activity of renalase. Additionally,a renalase inhibitor composition encompasses a chemically modifiedcompound, and derivatives, as is well known to one of skill in thechemical arts.

The renalase inhibitor compositions of the invention that decrease thelevel or activity (e.g., enzymatic activity, substrate binding activity,receptor binding activity, etc.) of renalase include antibodies, andfragments thereof. The antibodies of the invention include a variety offorms of antibodies including, for example, polyclonal antibodies,monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Faband F(ab)2, single chain antibodies (scFv), heavy chain antibodies (suchas camelid antibodies), synthetic antibodies, chimeric antibodies, and ahumanized antibodies. In one embodiment, the antibody of the inventionis an antibody that specifically binds to renalase. In some embodiments,the antibodies of the invention are bispecific antibodies, where thefirst specificity is to renalase and the second specificity is to atargeting molecule to guide the bispecific antibody to an anatomiclocation where the renalase binding is desired. In some embodiments, theantibodies of the invention are bispecific antibodies, where the firstspecificity is to renalase and the second specificity is to a secondbinding partner molecule that is carried and deployed to an anatomiclocation where renalase binding is desired.

Antibodies, including a renalase binding fragments thereof, of thepresent invention include, in certain embodiments, antibody amino acidsequences disclosed herein encoded by any suitable polynucleotide, orany isolated or formulated antibody. Further, antibodies of the presentdisclosure comprise antibodies having the structural and/or functionalfeatures of anti-renalase antibodies described herein. In oneembodiment, the anti-renalase antibody binds renalase and, therebypartially or substantially alters at least one biological activity ofrenalase (e.g., enzymatic activity, substrate binding activity, receptorbinding activity, etc.). In some embodiments, the renalase is humanrenalase.

In one embodiment, anti-renalase antibodies of the inventionimmunospecifically bind at least one specified epitope specific to therenalase protein, peptide, subunit, fragment, portion or any combinationthereof and do not specifically bind to other polypeptides, other thanrenalase from other species. The at least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofthe renalase protein. The term “epitope” as used herein refers to aprotein determinant capable of binding to an antibody. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

In some embodiments, the invention includes compositions comprising anantibody that specifically binds to renalase (e.g., binding portion ofan antibody). In one embodiment, the anti-renalase antibody is apolyclonal antibody. In another embodiment, the anti-renalase antibodyis a monoclonal antibody. In some embodiments, the anti-renalaseantibody is a chimeric antibody. In further embodiments, theanti-renalase antibody is a humanized antibody. In some embodiments, therenalase is human renalase. In some embodiments, the antibodies of theinvention specifically bind to at least one of SEQ ID NOS:1-7, 8, 50,92, 94, and fragments thereof. The binding portion of an antibodycomprises one or more fragments of an antibody that retain the abilityto specifically bind to binding partner molecule (e.g., renalase). Ithas been shown that the binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “binding portion” of an antibody include (i)a Fab fragment, a monovalent fragment consisting of the VL, VH, CL andCH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “binding portion” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.Binding portions can be produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins.

An antibody that binds to renalase of the invention is an antibody thatinhibits, blocks, or interferes with at least one renalase activity(e.g., enzymatic activity, substrate binding activity, receptor bindingactivity, etc.), in vitro, in situ and/or in vivo. A suitableanti-renalase antibody, specified portion, or variant can alsooptionally affect at least one renalase activity or function, such asbut not limited to, RNA, DNA or protein synthesis, protein release,renalase signaling, renalase cleavage, renalase activity, renalasereceptor binding, renalase production and/or synthesis.

In one embodiment, antibodies of the invention bind renalase. In oneembodiment, the antibodies specifically bind to renalase-1. In anotherembodiment, the antibodies specifically bind to renalase-2. In yetanother embodiment, the antibodies specifically bind to both renalase-1and renalase-2. In addition, epitope specific antibodies have beengenerated. Preferred antibodies of the invention include monoclonalantibodies 1C-22-1, 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7 and 3A-5-2.Examples of dual specificity antibodies, e.g. antibodies that recognizerenalase-1 and renalase-2 include antibodies 1C-22-1, 1D-28-4, 1D-37-10,and polyclonal antibodies as described in Table 1. Examples ofrenalase-type specific antibodies include 1F-26-1, 1F42-7, which arespecific for renalase-1. 3A-5-2 is specific for renalase-2. Sequencesencoding anti-renalase monoclonal antibodies are set forth in FIG. 5.

The nucleic acid (SEQ ID NO:52) and amino acid sequence (SEQ ID NO:9) ofthe heavy chain coding sequence of monoclonal antibody 1D-28-4 are foundin FIG. 5. The nucleic acid (SEQ ID NO:53) and amino acid sequence (SEQID NO:10) of the light chain coding sequence of monoclonal antibody1D-28-4 are found in FIG. 5.

The nucleic acid (SEQ ID NO:60) and amino acid sequence (SEQ ID NO:17)of the heavy chain coding sequence of monoclonal antibody 1D-37-10 arefound in FIG. 5. The nucleic acid (SEQ ID NO:61) and amino acid sequence(SEQ ID NO:18) of the light chain coding sequence of monoclonal antibody1D-37-10 are found in FIG. 5.

The nucleic acid (SEQ ID NO:68) and amino acid sequence (SEQ ID NO:25)of the heavy chain coding sequence of monoclonal antibody 1F-26-1 arefound in FIG. 5. The nucleic acid (SEQ ID NO:69) and amino acid sequence(SEQ ID NO:26) of the light chain coding sequence of monoclonal antibody1F-26-1 are found in FIG. 5.

The nucleic acid (SEQ ID NO:76) and amino acid sequence (SEQ ID NO:33)of the heavy chain coding sequence of monoclonal antibody 1F-42-7 arefound in FIG. 5.

The nucleic acid (SEQ ID NO:77) and amino acid sequence (SEQ ID NO:34)of the light chain coding sequence of monoclonal antibody 1F-42-7 arefound in FIG. 5.

The nucleic acid (SEQ ID NO:84) and amino acid sequence (SEQ ID NO:41)of the heavy chain coding sequence of monoclonal antibody 3A-5-2 arefound in FIG. 5. The nucleic acid (SEQ ID NO:85) and amino acid sequence(SEQ ID NO:42) of the light chain coding sequence of monoclonal antibody3A-5-2 are found in FIG. 5.

The underlined sequences in each of the sequences incorporate CDR1, CDR2and CDR3 sequences of each of the heavy and light chains.

Given that certain of the monoclonal antibodies can bind to the renalaseprotein, the VH and VL sequences can be “mixed and matched” to createother anti-renalase binding molecules of this disclosure. Renalasebinding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., immunoblot,Bia-Core, etc.). Preferably, when VH and VL chains are mixed andmatched, a VH sequence from a particular VH/VL pairing is replaced witha structurally similar VH sequence. Likewise, preferably a VL sequencefrom a particular VH/VL pairing is replaced with a structurally similarVL sequence.

Accordingly, in one aspect, this disclosure provides an isolatedmonoclonal antibody, or binding portion thereof comprising: (a) a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 9, 17, 25, 33 and 41; and (b) alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 10, 18, 26, 34 and 42, whereinthe antibody specifically binds a renalase protein.

Preferred heavy and light chain combinations include: (a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 9 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 10; or (b) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 18; or (c) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 25 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:26; or (d) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 33 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 34; or (e) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 41 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:42.

In another aspect, this disclosure provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 1D-28-4, 1D-37-10,1F-26-1, 1F42-7 or 3A-5-2, or combinations thereof. The amino acidsequences of the VH CDR1s of 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7 and3A-5-2 incorporate the sequences shown in SEQ ID NOs: 11, 19, 27, 35,and 43, respectively. The amino acid sequences of the VH CDR2s 1D-28-4,1D37-10, 1F-26-1, 1F42-7 or 3A-5-2 incorporate the sequences shown inSEQ ID NOs: 12, 20, 28, 36, and 44, respectively. The amino acidsequences of the VH CDR3s of 1D-28-4, 1D-3710, 1F-26-1, 1F42-7 or 3A-5-2incorporate the sequences shown in SEQ ID NOs: 13, 21, 29, 37, and 45,respectively. The amino acid sequences of the VK CDR1s of 1D-28-4,1D-37-10, 1F-26-1, 1F42-7 or 3A-5-2 incorporate the sequences shown inSEQ ID NOs: 14, 22, 30, 38, and 46, respectively. The amino acidsequences of the VK CDR2s of 1D-28-4, 1D-37-10, 1F26-1, 1F42-7 or 3A-5-2incorporate the sequences shown in SEQ ID NOs: 15, 23, 31, 39 and 47.The amino acid sequences of the VKCDR3s of 1D-28-4, 1D-37-10, 1F-26-1,1F42-7 or 3A-5-2 incorporate the sequences shown in SEQ ID NOs: 16, 24,32, 40 and 48, respectively. The CDR regions are delineated using theKabat system (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to renalase family membersand that binding specificity is provided primarily by the CDR1, CDR2,and CDR3 regions, the VH CDR1, CDR2, and CDR3 sequences and VL CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a VH CDR1, CDR2, and CDR3 and a VL CDR1, CDR2, and CDR3) tocreate other anti-renalase binding molecules of this disclosure.renalase binding of such “mixed and matched” antibodies can be testedusing the binding assays described above and in the Examples (e.g.,immunoblot, Biacore® analysis, etc). Preferably, when VH CDR sequencesare mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from aparticular VH sequence is replaced with a structurally similar CDRsequence(s). Likewise, when VL CDR sequences are mixed and matched, theCDR1, CDR2 and/or CDR3 sequence from a particular VL sequence preferablyis replaced with a structurally similar CDR sequence(s). It will bereadily apparent to the ordinarily skilled artisan that novel VH and VLsequences can be created by substituting one or more VH and/or VL CDRregion sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 1D-28-4, 1D-37-10,1F-26-1, 1F42-7 or 3A-5-2.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or binding portion thereof comprising at least oneselected from: (a) a heavy chain variable region CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:11, 19, 27, 35, and 43; (b) a heavy chain variable region CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 12, 20, 28, 36, and 44; (c) a heavy chain variable regionCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 13, 21, 29, 37, and 45; (d) a light chainvariable region CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 14, 22, 30, 38, and 46; (e) a lightchain variable region CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 15, 23, 31, 39 and 47; and (f)a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 24, 32, 40 and 48;wherein the antibody specifically binds an renalase.

In another embodiment, the antibody comprises at least one of the CDRsselected from: (a) a heavy chain variable region CDR1 comprising SEQ IDNO: 11; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 12;(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13; (d) alight chain variable region CDR1 comprising SEQ ID NO: 14; (e) a lightchain variable region CDR2 comprising SEQ ID NO: 15; and (f) a lightchain variable region CDR3 comprising SEQ ID NO: 16.

In another embodiment, the antibody comprises at least one of the CDRsselected from: (a) a heavy chain variable region CDR1 comprising SEQ IDNO: 19; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 20;(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 21; (d) alight chain variable region CDR1 comprising SEQ ID NO: 22; (e) a lightchain variable region CDR2 comprising SEQ ID NO: 23; and (f) a lightchain variable region CDR3 comprising SEQ ID NO: 24.

In another embodiment, the antibody comprises at least one of the CDRsselected from: (a) a heavy chain variable region CDR1 comprising SEQ IDNO: 27; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 28;(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29; (d) alight chain variable region CDR1 comprising SEQ ID NO: 30; (e) a lightchain variable region CDR2 comprising SEQ ID NO:31; and (f) a lightchain variable region CDR3 comprising SEQ ID NO: 32.

In another other embodiment, the antibody comprises at least one of theCDRs selected from: (a) a heavy chain variable region CDR1 comprisingSEQ ID NO: 35; (b) a heavy chain variable region CDR2 comprising SEQ IDNO: 36; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 37;(d) a light chain variable region CDR1 comprising SEQ ID NO: 38; (e) alight chain variable region CDR2 comprising SEQ ID NO: 39; and (f) alight chain variable region CDR3 comprising SEQ ID NO: 40.

In another embodiment, the antibody comprises at least one of the CDRsselected from: (a) a heavy chain variable region CDR1 comprising SEQ IDNO: 43; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 44;(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 45; (d) alight chain variable region CDR1 comprising SEQ ID NO: 46; (e) a lightchain variable region CDR2 comprising SEQ ID NO: 47; and (f) a lightchain variable region CDR3 comprising SEQ ID NO: 48.

The foregoing isolated anti-renalase antibody CDR sequences establish anovel family of renalase binding proteins, isolated in accordance withthis invention, and comprising polypeptides that include the CDRsequences listed. To generate and to select CDR's of the inventionhaving renalase binding and/or renalase detection and/or renalaseneutralization activity, standard methods known in the art forgenerating binding proteins of the present invention and assessing therenalase and/or renalase binding and/or detection and/or neutralizingcharacteristics of those binding protein may be used, including but notlimited to those specifically described herein.

Preferably, renalase binding molecules (e.g., antibodies, etc.) of thepresent invention, exhibit a high capacity to detect and bind renalasein a complex mixture of salts, compounds and other polypeptides, e.g.,as assessed by any one of several in vitro and in vivo assays known inthe art. The skilled artisan will understand that the renalase bindingmolecules (e.g., antibodies, etc.) described herein as useful in themethods of diagnosis and treatment and prevention of disease, are alsouseful in procedures and methods of the invention that include, but arenot limited to, an immunochromatography assay, an immunodot assay, aLuminex assay, an ELISA assay, an ELISPOT assay, a protein microarrayassay, a Western blot assay, a mass spectrophotometry assay, aradioimmunoassay (RIA), a radioimmunodiffusion assay, a liquidchromatography-tandem mass spectrometry assay, an ouchterlonyimmunodiffusion assay, reverse phase protein microarray, a rocketimmunoelectrophoresis assay, an immunohistostaining assay, animmunoprecipitation assay, a complement fixation assay, FACS, a proteinchip assay, separation and purification processes, and affinitychromatography (see also, 2007, Van Emon, Immunoassay and OtherBioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook,Gulf Professional Publishing; 1996, Diamandis and Christopoulos,Immunoassay, Academic Press; 2005, Joos, Microarrays in ClinicalDiagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today,John Wiley and Sons; 2007).

More preferably, the renalase binding molecules (e.g., antibodies, etc.)of the present invention, exhibit a high capacity to reduce or toneutralize renalase activity (e.g., enzymatic activity, substratebinding activity, receptor binding activity, etc.) as assessed by anyone of several in vitro and in vivo assays known in the art. Forexample, these renalase binding molecules (e.g., antibodies, etc.)neutralize renalase-associated or renalase-mediated disease or disorder.Preferably, renalase binding molecules (e.g., antibodies, etc.) of thepresent invention, also exhibit a high capacity to reduce or toneutralize renalase activity. In some embodiments, the renalase is humanrenalase.

As used herein, a renalase binding molecule (e.g., antibody, etc.) that“specifically binds to a renalase protein” is intended to refer to arenalase binding molecule (e.g., antibody, etc.) that binds to arenalase protein of any animal. In some embodiments, that antibody bindsto human renalase. Preferably, the a renalase binding molecule (e.g.,antibody, etc.) binds to a renalase protein with a KD of 1×10⁻⁶ M orless, more preferably 1×10⁻⁷ M or less, more preferably 1×10⁻⁸ M orless, more preferably 5×10⁻⁹ M or less, more preferably 1×10⁻⁹ M or lessor even more preferably 3×10⁻¹⁰ M or less. The term “does notsubstantially bind” to a protein or cells, as used herein, means doesnot bind or does not bind with a high affinity to the protein or cells,i.e., binds to the protein or cells with a KD of greater than 1×10⁶ M ormore, more preferably 1×10⁵ M or more, more preferably 1×10⁴ M or more,more preferably 1×10³ M or more, even more preferably 1×10² M or more.The term “KD”, as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) andis expressed as a molar concentration (M). KD values for a renalasebinding molecule (e.g., antibody, etc.) can be determined using methodswell established in the art. A preferred method for determining the KDof a binding molecule (e.g., antibody, etc.) is by using surface plasmonresonance, preferably using a biosensor system such as a Biacore®system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a KD of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M orless, even more preferably 1×10⁻⁸ M or less, even more preferably 5×10⁻⁹M or less and even more preferably 1×10⁻⁹ M or less for a target bindingpartner molecule. However, “high affinity” binding can vary for otherantibody isotypes. For example, “high affinity” binding for an IgMisotype refers to an antibody having a KD of 10⁻⁶M or less, morepreferably 10⁻⁷M or less, even more preferably 10⁻⁸M or less.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Generation Of Anti-Renalase Antibodies

The invention provides compositions that bind to renalase. The renalasemolecules disclosed herein are a class of molecules that include thosehaving high and/or significant sequence identity with other polypeptidesdisclosed herein. More specifically, the putative renalase will share atleast about 40% sequence identity with a nucleic acid having thesequence SEQ ID NO: 49 or 51. More preferably, a nucleic acid encodingrenalase has at least about 45% identity, or at least about 50%identity, or at least about 55% identity, or at least about 60%identity, or at least about 65% identity, or at least about 70%identity, or at least about 75% identity, or at least about 80%identity, or at least about 85% identity, or at least about 90%identity, or at least about 95% identity, or at least about 98%, or atleast about 99% sequence identity with SEQ ID NO:49 or 51 disclosedherein. Even more preferably, the nucleic acid is SEQ ID NO:49 or 51 or93 or 95. The term “renalase” also includes renalase isoforms. Therenalase gene contains 9 exons spanning 310188 bp in chromosome 10 ofhuman genome. The renalase clone (SEQ ID NO: 49, GenBank accessionnumber: BC005364) disclosed herein is the gene containing exons 1, 2, 3,4, 5, 6, 8. There are at least two additional alternatively-splicedforms of renalase protein as shown in the human genome database. Onealternatively spliced form contains exons 1, 2, 3, 4, 5, 6, 9,identified by clones in the human genome database as GenBank accessionnumber AK002080 and NMJ)18363, the sequences of which are expresslyincorporated herein by reference. The other alternatively spliced formcontains exons 5, 6, 7, 8, identified by clones in the human genomedatabase as GenBank accession number BX648154, the sequence of which isexpressly incorproated herein by reference. Unless otherwise indicated,“renalase” encompasses all known renalases (e.g., rat renalase, andhuman renalase), and renalases to be discovered, including but notlimited to, human renalase and chimpanzee renalase, having thecharacteristics and/or physical features of the renalase disclosedherein.

In addition, the putative renalase share at least about 60% sequenceidentity with a polypeptide having the sequence SEQ ID NO:8 or 50. Morepreferably, renalase has at least about 45% identity, or at least about50% identity, or at least about 55% identity, or at least about 60%identity, or at least about 65% identity, or at least about 70%identity, or at least about 75% identity, or at least about 80%identity, or at least about 85% identity, or at least about 90%identity, or at least about 95% identity, or at least about 98%, or atleast about 99% sequence identity with SEQ ID NO:8 or 50 disclosedherein. Even more preferably, the renalase polypeptide has the aminoacid sequence of SEQ ID NO:8 or 50 or 92 or 94.

In one embodiment, the antibodies of the invention can be generated byusing a peptide derived from the sequence of renalase to immunize ananimal whereby the animal produces antibodies directed against theimmunogen. Exemplary immunogens include peptide derived from renalase.That is, peptides having fragments of the renalase sequence can be usedin the inventions. Peptides can be produced in a variety of ways,including expression as recombinant peptides, expression as largerpolypeptides and cleaved enzymatically or chemically. Alternatively,they may be produced synthetically as is known in the art. Preferredpeptides as used to generate affinity reagents of the present inventionare found in Table 1 (SEQ ID NOs: 1-7).

Anti-renalase antibodies of the present invention can be optionallyproduced by a variety of techniques, including the standard somatic cellhybridization technique (hybridoma method) of Kohler and Milstein (1975)Nature 256:495. In the hybridoma method, a mouse or other appropriatehost animal, such as a hamster or macaque monkey, is immunized asdescribed herein to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse or rabbit or other species immunizedwith polypeptide or peptide of the invention with myeloma cells and thenscreening the hybridomas resulting from the fusion for hybridoma clonesthat secrete an antibody able to bind a polypeptide of the invention.Briefly, mice can be immunized with a renalase polypeptide or peptidethereof. In a preferred embodiment, the renalase polypeptide or peptidethereof is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks.

Alternatively, rabbits can be immunized with a renalase polypeptide orpeptide thereof. In this embodiment, either full length renalaseproteins or peptides derived from renalase can be used as immunogens.

Renalase used in the invention can take a variety of forms. For example,they can include purified renalase proteins or fragments thereof,recombinantly produced renalase or fragments thereof. In someembodiments, the renalase is human renalase. When recombinant renalaseis used, it can be produced in eukaryotic or prokaryotic cells as isknown in the art. Additional immunogens include peptides derived fromrenalase. That is, peptides having fragments of the renalase sequencecan be used in the inventions. Peptides can be produced in a variety ofways, including expression as recombinant peptides, expression as largerpolypeptides and cleaved enzymatically or chemically. Alternatively,they may be produced synthetically as is known in the art. Preferredpeptides as used to generate affinity reagents of the present inventionare found in Table 1 (SEQ ID NOs: 1-7). The full-length amino acidsequence of human renalase is depicted in SEQ ID NO:8, where a knownpolymorphism is possible as indicated (compare to SEQ ID NO. 92). Theamino acid sequence of renalase-2 is found in SEQ ID NO:50, again wherea known polymorphism is possible as indicated (compare to SEQ ID NO.94). It is appreciated that other polymorphisms exist. These also areincluded in the definition of renalase. In some embodiments, therenalase binding molecules of the invention specifically bind to atleast one of SEQ ID NOS:1-7, 8, 50, 92, 94, and fragments thereof.

The anti-renalase antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-renalase antibody can be isolated from suchanimals and immortalized using suitable methods, such as the methodsdescribed herein. Alternatively, the antibody coding sequences may becloned, introduced into a suitable vector, and used to transfect a hostcell for expression and isolation of the antibody by methods taughtherein and those known in the art.

The use of transgenic mice carrying human immunoglobulin (Ig) loci intheir germline configuration provide for the isolation of high affinityfully human monoclonal antibodies directed against a variety of targetsincluding human self antigens for which the normal human immune systemis tolerant (Lonberg, N. et al., U.S. Pat. No. 5,569,825, U.S. Pat. No.6,300,129 and 1994, Nature 368:856-9; Green, L. et al., 1994, NatureGenet. 7:13-21; Green, L. & Jakobovits, 1998, Exp. Med. 188:483-95;Lonberg, N. and Huszar, D., 1995, Int. Rev. Immunol. 13:65-93;Kucherlapati, et al. U.S. Pat. No. 6,713,610; Bruggemann, M. et al.,1991, Eur. J. Immunol. 21:1323-1326; Fishwild, D. et al., 1996, Nat.Biotechnol. 14:845-851; Mendez, M. et al., 1997, Nat. Genet. 15:146-156;Green, L., 1999, J. Immunol. Methods 231:11-23; Yang, X. et al., 1999,Cancer Res. 59:1236-1243; Bruggemann, M. and Taussig, M J., Curr. Opin.Biotechnol. 8:455-458, 1997; Tomizuka et al. WO02043478). The endogenousimmunoglobulin loci in such mice can be disrupted or deleted toeliminate the capacity of the animal to produce antibodies encoded byendogenous genes. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Medarex (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected target bindingpartner molecule (e.g., antigen, etc.) using technology as describedelsewhere herein.

In another embodiment, the human antibody is selected from a phagelibrary, where that phage comprises human immunoglobulin genes and thelibrary expresses human antibody binding domains as, for example, singlechain antibodies (scFv), as Fab, or some other construct exhibitingpaired or unpaired antibody variable regions (Vaughan et lo al. NatureBiotechnology 14:309-314 (1996): Sheets et al. PITAS (USA) 95:6157-6162(1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks etal. J. Mol. Biol., 222:581 (1991)). Human monoclonal antibodies of theinvention can also be prepared using phage display methods for screeninglibraries of human immunoglobulin genes. Such phage display methods forisolating human antibodies are established in the art. See for example:U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No. 5,571,698 toLadner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.;U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S.Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and6,593,081 to Griffiths et al.

Preparation of immunogenic antigens, and monoclonal antibody productioncan be performed using any suitable technique such as recombinantprotein production. The immunogenic antigens can be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen can be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art. DNAencoding the monoclonal antibodies is readily isolated and sequencedusing methods known in the art (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). Where a hybridoma is produced, suchcells can serve as a source of such DNA. Alternatively, using displaytechniques wherein the coding sequence and the translation product arelinked, such as phage or ribosomal display libraries, the selection ofthe binder and the nucleic acid is simplified. After phage selection,the antibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired binding fragment, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria.

Humanized Antibodies

The invention further provides humanized immunoglobulins (or antibodies)which bind human renalase. The humanized forms of immunoglobulins havevariable framework region(s) substantially from a human immunoglobulin(termed an acceptor immunoglobulin) and CDRs substantially from anon-human mAbs which specifically binds renalase. The constantregion(s), if present, are also substantially from a humanimmunoglobulin. The humanized antibodies exhibit K_(D) for renalase ofat least about 10⁻⁶M (1 microM), about 10⁻⁷M (100 nM), or less. Thebinding affinity of the humanized antibodies may be greater or less thanthat of the mouse antibody from which they were derived. To affect achange in affinity, improve affinity, of the humanized antibody forrenalase substitutions in either the CDR residues or the human residuesmay be made. The source for production of humanized antibody which bindsto renalase is preferably the 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7 or3A-5-2 mouse antibodies whose generation, isolation and characterizationare described in the Examples provided herein, although other mouseantibodies, which compete with the 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7 or3A-5-2 mouse antibodies for binding to renalase can also be used. Theidentified CDRs set forth in the sequece listing can be a starting pointof the humanization process. For example, any one or more of thefollowing amino acid sequences (and corresponding nucleic acid sequencesthereof) can be a starting point of the humanization process: (a) aheavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 19, 27, 35, and43; (b) a heavy chain variable region CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 12, 20, 28,36, and 44; (c) a heavy chain variable region CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 13, 21,29, 37, and 45; (d) a light chain variable region CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:14, 22, 30, 38, and 46; (e) a light chain variable region CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 15, 23, 31, 39 and 47; and (f) a light chain variable regionCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16, 24, 32, 40 and 48.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies, be derived from human germlineimmunoglobulin sequences, or can be consensus sequences of several humanantibody and/or germline sequences.

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

In one example, the amino acid sequence of anti-renalase mAb is used toquery a human antibody database compiled from public antibody sequencedatabases. The heavy chain variable region can be used to find the humanvariable region with the highest sequence identity. The variable regionof the light chain can, similarly, be used to find the human variableregion with the highest sequence identity. A DNA construct in which theregions coding for the CDRs of one of the heavy chain variable regionsfrom the murine mAbs donor are transferred into the selected human heavychain variable sequence, replacing the CDRs of the human variable regionis prepared for each murine variable region.

The unnatural juxtaposition of murine CDR regions with human variableframework region can result in unnatural conformational restraints,which, unless corrected by substitution of certain amino acid residues,lead to loss of binding affinity. As noted supra, the humanizedantibodies of the invention comprise variable framework region(s)substantially from a human immunoglobulin and CDRs substantially from amouse immunoglobulin. Having identified the CDRs of mouse antibodies andappropriate human acceptor immunoglobulin sequences, the next step is todetermine which, if any, residues from these components should besubstituted to optimize the properties of the resulting humanizedantibody. In general, substitution of human amino acid residues withmurine should be minimized, because introduction of murine residuesincreases the risk of the antibody eliciting a HAMA response in humans.Amino acids are selected for substitution based on their possibleinfluence on CDR conformation and/or binding to the target bindingpartner molecule. Investigation of such possible influences can be doneby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids. With regard tothe empirical method, it has been found to be particularly convenient tocreate a library of variant sequences that can be screened for thedesired activity, binding affinity or specificity. One format forcreation of such a library of variants is a phage display vector.Alternatively, variants can be generated using other methods forvarigation of a nucleic acid sequence encoding the targeted residueswithin the variable domain.

Another method of determining whether further substitutions arerequired, and the selection of amino acid residues for substitution, canbe accomplished using computer modeling. Computer hardware and softwarefor producing three-dimensional images of immunoglobulin molecules arewidely available. In general, molecular models are produced startingfrom solved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsin the mouse antibody from which they were derived. Although not usuallydesirable, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin.Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin.

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de nova solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide. All nucleic acids encoding the antibodiesdescribed in this application are expressly included in the invention.

The variable segments of humanized antibodies produced as describedsupra are typically linked to at least a portion of a humanimmunoglobulin constant region. The antibody will contain both lightchain and heavy chain constant regions. The heavy chain constant regionusually includes CH1, hinge, CH2, CH3, and, sometimes, CH4 domains. Thehumanized antibodies may comprise any type of constant domains from anyclass of antibody, including IgM, IgG, IgD, IgA and IgE, and anysubclass (isotype), including IgG1, IgG2, IgG3 and IgG4. When it isdesired that the humanized antibody exhibit cytotoxic activity, theconstant domain is usually a complement-fixing constant domain and theclass is typically IgG₁. When such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype.

Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include a signal sequence, a promoter, anenhancer, and a transcription termination sequence (see Queen et al.,Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.Immunol. 148, 1149 (1992), which are incorporated herein by reference intheir entirety for all purposes).

Methods of Using the Renalase Binding Molecules

Given the properties of the renalase binding molecules (e.g.,antibodies, etc.) of the present invention, the renalase bindingmolecules are suitable as diagnostic, therapeutic and prophylacticagents for diagnosing, treating or preventing renalase-associatedconditions in humans and animals.

In general, use comprises administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesor binding fragments of the present invention to a susceptible subjector one exhibiting a condition in which renalase activity is known tohave pathological sequelae, such as tumor growth and metastasis. Anyactive form of the renalase binding molecule can be administered,including antibody Fab and F(ab′)2 fragments.

Preferably, the renalase binding molecule used is compatible with therecipient species such that the immune response to the renalase bindingmolecule does not result in an unacceptably short circulating half-lifeor induce an immune response to the renalase binding molecule in thesubject. Preferably, the renalase binding molecule administered exhibitssome secondary functions such as binding to Fc receptors of the subjectand activation of ADCC mechanisms.

Treatment of individuals may comprise the administration of atherapeutically effective amount of the renalase binding molecules ofthe present invention. The renalase binding molecules can be provided ina kit as described below. The renalase binding molecules can be used oradministered as a mixture, for example in equal amounts, orindividually, provided in sequence, or administered all at once. Inproviding a patient with renalase binding molecule, the dosage ofadministered agent will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition, previousmedical history, etc.

In general, if administering a systemic dose of a renalase bindingmolecule, it is desirable to provide the recipient with a dosage of arenalase binding molecule which is in the range of from about 1ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500 ng/kg-1 μg/kg, 1 μg/kg-100μg/kg, 100 μg/kg-500 μg/kg, 500 μg/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg (body weight of recipient),although a lower or higher dosage may be administered. Dosages as low asabout 1.0 mg/kg may be expected to show some efficacy. Preferably, about5 mg/kg is an acceptable dosage, although dosage levels up to about 50mg/kg are also preferred especially for therapeutic use. Alternatively,administration of a specific amount of the renalase binding molecule maybe given which is not based upon the weight of the patient such as anamount in the range of 1 μg-100 μg, 1 mg-100 mg, or 1 gm-100 gm. Forexample, site specific administration may be to body compartment orcavity such as intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,vaginal, rectal, buccal, sublingual, intranasal, ophthalmic, ortransdermal means.

The renalase binding molecule composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or ophthalmically such as, but not limited to, eye drops; or forthe treatment of dental disease; or transdermally such as not limited toa gel, ointment, lotion, suspension or patch delivery system withchemical enhancers such as dimethyl sulfoxide to either modify the skinstructure or to increase the drug concentration in the transdermalpatch, or with oxidizing agents that enable the application offormulations containing proteins and peptides onto the skin (WO98/53847), or applications of electric fields to create transienttransport pathways such as electroporation, or to increase the mobilityof charged drugs through the skin such as iontophoresis, or applicationof ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and4,767,402).

In a similar approach, another therapeutic use of the renalase bindingmolecule of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-renalaseresponse (Linthicum, D. S, and Farid, N. R., Anti-Idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300).

The renalase binding molecules of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A. ed., MackEaston Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the above-described compoundstogether with a suitable amount of carrier vehicle. Additionalpharmaceutical methods may be employed to control the duration ofaction. Controlled release preparations may be achieved through the useof polymers to complex or absorb the compounds. Another possible methodto control the duration of action by controlled release preparations isto incorporate the compounds of the present invention into particles ofa polymeric material such as polyesters, polyamino acids, hydrogels,poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, interfacial polymerization, for example, hydroxymethylcelluloseor gelatin-microcapsules and poly(methylmethacylate)-microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The present invention also provides kits which are useful for carryingout the present invention. The present kits comprise a first containercontaining or packaged in association with the above-describedantibodies. The kit may also comprise another container containing orpackaged in association solutions necessary or convenient for carryingout the invention. The containers can be made of glass, plastic or foiland can be a vial, bottle, pouch, tube, bag, etc. The kit may alsocontain written information, such as procedures for carrying out thepresent invention or analytical information, such as the amount ofreagent contained in the first container means. The container may be inanother container apparatus, e.g. a box or a bag, along with the writteninformation.

Yet another aspect of the present invention is a kit for detectingrenalase in a biological sample. The kit includes a container holdingone or more renalase binding molecules which binds an epitope ofrenalase and instructions for using the renalase binding molecule forthe purpose of binding to renalase to form complex and detecting theformation of the complex such that the presence or absence of thecomplex correlates with presence or absence of renalase in the sample.Examples of containers include multiwell plates which allow simultaneousdetection of renalase in multiple samples.

Combination Therapy

The renalase binding molecule compositions of the invention can be usedin combination with another therapeutic treatment or agent to treat thedisease or disorder. For example, the renalase binding molecule of theinvention may be administered alone, or in combination with one or moretherapeutically effective agents or treatments. The othertherapeutically effective agent may be conjugated to the renalasebinding molecule of the invention, incorporated into the samecomposition as the renalase binding molecule of the invention, or may beadministered as a separate composition. The other therapeutically agentor treatment may be administered prior to, during and/or after theadministration of the antibody of the invention or related compound.

In certain embodiments, the renalase binding molecule of the inventionis co-administered with one or more other therapeutic agents ortreatments. In other embodiments, the renalase binding molecule of theinvention is administered independently from the administration of oneor more other therapeutic agents or treatments. For example, therenalase binding molecule of the invention is administered first,followed by the administration of one or more other therapeutic agentsor treatments. Alternatively, one or more other therapeutic agents areadministered first, followed by the administration of a renalase bindingmolecule of the invention. As another example, a treatment (e.g., asurgery, radiation, etc.) is carried out first, followed by theadministration of the renalase binding molecule of the invention.

Other therapeutically effective agents/treatments include surgery,anti-neoplastics (including chemotherapeutic agents and radiation),anti-angiogenesis agents, antibodies to other targets, small molecules,photodynamic therapy, immunotherapy, immunity enhancing therapy,cytotoxic agents, cytokines, chemokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, cardioprotectants,immunostimulatory agents, immunosuppressive agents, and agents thatpromote proliferation of hematological cells.

In one embodiment, the “another therapeutic agent,” as used herein, aresecond, distinct therapeutic agents or anti-cancer agents, i.e.,therapeutic agents or anti-cancer agents “other than” the renalasebinding molecule of the invention. Any secondary therapeutic agent maybe used in the combination therapies of the present invention. Also,secondary therapeutic agents or “second anti-cancer agents” may beselected with a view to achieving additive, greater than additive andpotentially synergistic effects, according to the following guidance.

To practice combined anti-tumor therapy, one would administer to ananimal or patient a renalase binding molecule of the invention incombination with another, i.e., a second, distinct anti-cancer agent ina manner effective to result in their combined anti-tumor actions withinthe animal or patient. The agents would therefore be provided in amountseffective and for periods of time effective to result in their combined,or concurrent, presence within the tumor or tumor vasculature and theircombined actions in the tumor environment. To achieve this goal, therenalase binding molecule of the invention and the second, distinctanti-cancer agents may be administered to the animal substantiallysimultaneously, either in a single composition, or as two distinctcompositions using different administration routes.

Alternatively, the renalase binding molecule of the invention mayprecede, or follow, the second, distinct anti-cancer agent by, e.g.,intervals ranging from minutes to weeks. In certain embodiments wherethe renalase binding molecule of the invention and the second, distinctanti-cancer agents are applied separately to the animal, one wouldensure that a significant period of time did not expire between the timeof each delivery, such that each agent would still be able to exert anadvantageously combined effect on the tumor. In such instances, it iscontemplated that one would contact the tumor with both agents withinabout 5 minutes to about one week of each other and, more preferably,within about 12-72 hours of each other, with a delay time of only about12-48 hours being most preferred.

The secondary therapeutic agents for separately timed combinationtherapies may be selected based upon certain criteria, including thosediscussed elsewhere herein. However, a preference for selecting one ormore second, distinct anti-cancer agents for prior or subsequentadministration does not preclude their use in substantially simultaneousadministration if desired. Second, distinct anti-cancer agents selectedfor administration “prior to” the primary therapeutic agents of thepresent invention, and designed to achieve increased and potentiallysynergistic effects.

Second, distinct anti-cancer agents selected for administration“subsequent to” the primary therapeutic agents of the present invention,and designed to achieve increased and potentially synergistic effects,include agents that benefit from the effects of the primary therapeuticagent. Accordingly, effective second, distinct anti-cancer agents forsubsequent administration include anti-angiogenic agents, which inhibitmetastasis; agents targeting necrotic tumor cells, such as antibodiesspecific for intracellular binding partner molecules that becomeaccessible from malignant cells in vivo (U.S. Pat. Nos. 5,019,368,4,861,581 and 5,882,626, each specifically incorporated herein byreference); chemotherapeutic agents; and anti-tumor cellimmunoconjugates, which attack any tumor cells.

The renalase binding molecule of the invention can also be administeredin combination with a cancer immunotherapy. The cancer immunotherapy canbe one designed to elicit a humoral immune response against thesubject's cancer cells, or a cell-mediated immune response against thesubject's cancer cells, or a combination of a humoral response and acell-mediated response against the subject's cancer cells. Non-limitingexamples of cancer immunotherapy useful in combination with the renalasebinding molecules of the invention include a cancer vaccine, a DNAcancer vaccine, adoptive cellular therapy, adoptive immunotherapy, CART-cell therapy, antibodies, immunity enhancing compounds, cytokines,interleukins (e.g., IL-2, etc.), interferons (IFN-α, etc.), andcheckpoint inhibitors (e.g., PD-1 inhibitor, CTLA-4 inhibitor, etc.).

In some situations, it may be desirable to extend the time period fortreatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as the primary therapeuticagent of the present invention, and another treatment was intended toprevent micrometastasis or tumor re-growth, such as the administrationof an anti-angiogenic agent. Anti-angiogenics should be administered ata careful time after surgery, however, to allow effective wound healing.Anti-angiogenic agents may then be administered for the lifetime of thepatient.

It is also envisioned that more than one administration of either therenalase binding molecule of the invention or the second, distinctanti-cancer agent will be utilized. The renalase binding molecule of theinvention and the second, distinct anti-cancer agent may be administeredinterchangeably, on alternate days or weeks; or a sequence of one agenttreatment may be given, followed by a sequence of the other treatment.In any event, to achieve tumor regression using a combined therapy, allthat is required is to deliver both agents in a combined amounteffective to exert an anti-tumor effect, irrespective of the times foradministration.

Chemotherapeutic drugs can be used in combination with the renalaseinhibitors of the invention. Chemotherapeutic drugs can killproliferating tumor cells, enhancing the necrotic areas created by theoverall treatment.

One aspect of the invention provides a method of treating or preventingcancer using a renalase inhibitor of the invention. The skilled artisanwill understand that treating or preventing cancer in a patientincludes, by way of non-limiting examples, killing and destroying acancer cell, as well as reducing the proliferation of or cell divisionrate of a cancer cell. The skilled artisan will also understand that acancer cell can be, by way of non-limiting examples, a primary cancercell, a cancer stem cell, a metastatic cancer cell. The following arenon-limiting examples of cancers that can be treated by the disclosedmethods and compositions: Acute Lymphoblastic; Acute Myeloid Leukemia;Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AppendixCancer; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; BladderCancer; Bone Cancer; Osteosarcoma and Malignant Fibrous Histiocytoma;Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, BrainStem Glioma, Childhood; Brain Tumor, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Childhood; Central Nervous System EmbryonalTumors; Cerebellar Astrocytoma; Cerebral Astrocytotna/Malignant Glioma;Craniopharyngioma; Ependymoblastoma; Ependymoma; Medulloblastoma;Medulloepithelioma; Pineal Parenchymal Tumors of intermediateDifferentiation; Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma; Visual Pathway and Hypothalamic Glioma; Brain and SpinalCord Tumors; Breast Cancer; Bronchial Tumors; Burkitt Lymphoma;Carcinoid Tumor; Carcinoid Tumor, Gastrointestinal; Central NervousSystem Atypical Teratoid/Rhabdoid Tumor; Central Nervous SystemEmbryonal Tumors; Central Nervous System Lymphoma; CerebellarAstrocytoma Cerebral Astrocytoma/Malignant Glioma, Childhood; CervicalCancer; Chordoma, Childhood; Chronic Lymphocytic Leukemia; ChronicMyelogenous Leukemia; Chronic Myeloproliferative Disorders; ColonCancer; Colorectal Cancer; Craniopharyngioma; Cutaneous T-Cell Lymphoma;Esophageal Cancer; Ewing Family of Tumors; Extragonadal Germ Cell Tumor;Extrahepatic Bile Duct Cancer; Eye Cancer, intraocular Melanoma; EyeCancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumor (GIST);Germ Cell Tumor, Extracranial; Germ Cell Tumor, Extragonadal; Germ CellTumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma,Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma,Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head andNeck Cancer; Hepatocellular (Liver) Cancer; Histiocytosis, LangerhansCell; Hodgkin Lymphoma; Hypopharyngeal Cancer; Hypothalamic and VisualPathway Glioma; intraocular Melanoma; Islet Cell Tumors; Kidney (RenalCell) Cancer; Langerhans Cell Histiocytosis; Laryngeal Cancer; Leukemia,Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, ChronicLymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lipand Oral Cavity Cancer; Liver Cancer; Lung Cancer, Non-Small Cell; LungCancer, Small Cell; Lymphoma, AIDS-Related; Lymphoma, Burkitt; Lymphoma,Cutaneous T-Cell; Lymphoma, Hodgkin; Lymphoma, Non-Hodgkin; Lymphoma,Primary Central Nervous System; Macroglobulinemia, Waldenstrom;Malignant Fibrous Histiocvtoma of Bone and Osteosarcoma;Medulloblastoma; Melanoma; Melanoma, intraocular (Eye); Merkel CellCarcinoma; Mesothelioma; Metastatic Squamous Neck Cancer with OccultPrimary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome,(Childhood); Multiple Myeloma/Plasma Cell Neoplasm; Mycosis; Fungoides;Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases;Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Neuroblastoma; Non-Small Cell Lung Cancer; OralCancer; Oral Cavity Cancer; Oropharyngeal Cancer; Osteosarcoma andMalignant Fibrous Histiocytoma of Bone; Ovarian Cancer; OvarianEpithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low MalignantPotential Tumor; Pancreatic Cancer; Pancreatic Cancer, Islet CellTumors; Papillomatosis; Parathyroid Cancer; Penile Cancer; PharyngealCancer; Pheochromocytoma; Paraganglioma; Pineal Parenchymal Tumors ofIntermediate Differentiation; Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors; Pituitary Tumor; Plasma Celt Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Primary Central Nervous SystemLymphoma; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer;Renal Pelvis and Ureter, Transitional Cell Cancer; Respiratory TractCarcinoma Involving the NUT Gene on Chromosome 15; Retinoblastoma;Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing Family ofTumors; Sarcoma, Kaposi; Sarcoma, Soft Tissue; Sarcoma, Uterine; SezarySyndrome; Skin Cancer (Nonmelanoma); Skin Cancer (Melanoma); SkinCarcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer;Soft Tissue Sarcoma; Squamous Cell Carcinoma, Squamous Neck Cancer withOccult Primary, Metastatic; Stomach (Gastric) Cancer; SupratentorialPrimitive Neuroectodermal Tumors; T-Cell Lymphoma, Cutaneous; TesticularCancer; Throat Cancer; Thymoma and Thymic Carcinoma; Thyroid Cancer;Transitional Cell Cancer of the Renal Pelvis and Ureter; TrophoblasticTumor, Gestational; Urethral Cancer; Uterine Cancer, Endometrial;Uterine Sarcoma; Vaginal Cancer; Vulvar Cancer; WaldenstromMacroglobulinemia; and Wilms Tumor.

In one embodiment, the invention provides a method to treat cancercomprising treating the subject prior to, concurrently with, orsubsequently to the administration of the renalase binding molecule ofthe invention, with a complementary therapy for the cancer, such assurgery, chemotherapy, chemotherapeutic agent, radiation therapy, orhormonal therapy or a combination thereof.

Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil,cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine(CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium,altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan,cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferonalfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxicalkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide,melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley,AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU,CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone,cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide,dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide,melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard,PCNU, piperazine, piperazinedione, pipobroman, porfiromycin,spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin,thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864),antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine,colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxelderivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastinesulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D,bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristinesulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26,navelbine and taxotere), biologicals (e.g., alpha interferon, BCG,G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g.,camptothecin, camptothecin derivatives, and morpholinodoxorubicin),topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA,anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL,daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin,oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g.,hydroxyurea, procarbazine, o,p′-DDD, dacarbazine, CCNU, BCNU,cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole,hexamethylmelamine, all-trans retinoic acid, gliadel and porfimersodium).

Antiproliferative agents are compounds that decrease the proliferationof cells. Antiproliferative agents include alkylating agents,antimetabolites, enzymes, biological response modifiers, miscellaneousagents, hormones and antagonists, androgen inhibitors (e.g., flutamideand leuprolide acetate), antiestrogens (e.g., tamoxifen citrate andanalogs thereof, toremifene, droloxifene and roloxifene), Additionalexamples of specific antiproliferative agents include, but are notlimited to levamisole, gallium nitrate, granisetron, sargramostimstrontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, andondansetron.

The renalase binding molecule of the invention can be administered aloneor in combination with other anti-tumor agents, includingcytotoxic/antineoplastic agents and anti-angiogenic agents.Cytotoxic/anti-neoplastic agents are defined as agents which attack andkill cancer cells. Some cytotoxic/anti-neoplastic agents are alkylatingagents, which alkylate the genetic material in tumor cells, e.g.,cis-platin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracilmustard, chlomaphazin, and dacabazine. Other cytotoxic/anti-neoplasticagents are antimetabolites for tumor cells, e.g., cytosine arabinoside,fluorouracil, methotrexate, mercaptopuirine, azathioprime, andprocarbazine. Other cytotoxic/anti-neoplastic agents are antibiotics,e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin,mitomycin, mytomycin C, and daunomycin. There are numerous liposomalformulations commercially available for these compounds. Still othercytotoxic/anti-neoplastic agents are mitotic inhibitors (vincaalkaloids). These include vincristine, vinblastine and etoposide.Miscellaneous cytotoxic/anti-neoplastic agents include taxol and itsderivatives, L-asparaginase, anti-tumor antibodies, dacarbazine,azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, andvindesine.

Anti-angiogenic agents are well known to those of skill in the art.Suitable anti-angiogenic agents for use in the methods and compositionsof the present disclosure include anti-VEGF antibodies, includinghumanized and chimeric antibodies, anti-VEGF aptamers and antisenseoligonucleotides. Other known inhibitors of angiogenesis includeangiostatin, endostatin, interferons, interleukin 1 (including alpha andbeta) interleukin 12, retinoic acid, and tissue inhibitors ofmetalloproteinase-1 and -2. (TIMP-1 and -2). Small molecules, includingtopoisomerases such as razoxane, a topoisomerase II inhibitor withanti-angiogenic activity, can also be used.

Other anti-cancer agents that can be used in combination with thedisclosed compounds include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; albumin-bound paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine; vinorelbine tartrate; vinrosidinesulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin;zorubicin hydrochloride. Other anti-cancer drugs include, but are notlimited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil;abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+ progesterone; leuprorelin; levamisole; liarozole; linearpolyamine analogue; lipophilic disaccharide peptide; lipophilic platinumcompounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol;lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+ pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; imilimumab;mirtazapine; BrUOG 278; BrUOG 292; RAD0001; CT-011; folfirinox;tipifarnib; R115777; LDE225; calcitriol; AZD6244; AMG 655; AMG 479;BKM120; mFOLFOX6; NC-6004; cetuximab; IM-C225; LGX818; MEK162; BBI608;MEDI4736; vemurafenib; ipilimumab; ivolumab; nivolumab; panobinostat;leflunomide; CEP-32496; alemtuzumab; bevacizumab; ofatumumab;panitumumab; pembrolizumab; rituximab; trastuzumab; STAT3 inhibitors(e.g., STA-21, LLL-3, LLL12, XZH-5, S31-201, SF-1066, SF-1087, STX-0119,cryptotanshinone, curcumin, diferuloylmethane, FLLL11, FLLL12, FLLL32,FLLL62, C3, C30, C188, C188-9, LYS, OPB-31121, pyrimethamine, OPB-51602,AZD9150, etc.); hypoxia inducing factor 1 (HIF-1) inhibitors (e.g., LW6,digoxin, laurenditerpenol, PX-478, RX-0047, vitexin, KC7F2, YC-1, etc.)and zinostatin stimalamer. In one embodiment, the anti-cancer drug is5-fluorouracil, taxol, or leucovorin.

Methods of Diagnosis

In some embodiments, an increase in the level of renalase, or a renalasefragment, in a subject's cell, tissue, or bodily fluid, compared with acomparator is used in the methods of the invention as marker for thediagnosis of a disease or disorder, assessing the severity of a diseaseor disorder, and for monitoring the effect or effectiveness of atreatment of a disease or disorder. In various embodiments, the diseaseor disorder is acute renal failure (i.e., acute tubular necrosis, orATN, an ischemic condition in the kidney), a cardiovascular disease ordisorder (e.g., hypertension, pulmonary hypertension, systolichypertension, diabetic hypertension, asymptomatic left ventriculardysfunction, chronic congestive heart failure, myocardial infarction,cardiac rhythm disturbance, atherosclerosis, etc.), cancer, heartdisease or disorder, a kidney disease or disorder, a gastrointestinaldisease or disorder, a liver disease or disorder, a lung disease ordisorder, a pancreas disease or disorder (e.g., pancreatitis), mentaldisease or disorder (e.g., depression, anxiety, etc.), or a neurologicaldisease or disorder.

In one embodiment, the invention is a method of diagnosing a disease ordisorder of a subject by assessing the level of renalase, or a renalasefragment, in a biological sample of the subject. In one embodiment, thebiological sample of the subject is a cell, tissue, or bodily fluid.Non-limiting examples of bodily fluids in which the level of renalase,or a renalase fragment, can be assessed include, but are not limited to,blood, serum, plasma and urine. In various embodiments, the level ofrenalase, or a renalase fragment, in the biological sample of thesubject is compared with the renalase, or the renalase fragment, levelin a comparator. Non-limiting examples of comparators include, but arenot limited to, a negative control, a positive control, an expectednormal background value of the subject, a historical normal backgroundvalue of the subject, an expected normal background value of apopulation that the subject is a member of, or a historical normalbackground value of a population that the subject is a member of. Invarious embodiments, the disease or disorder is acute renal failure(i.e., acute tubular necrosis, or ATN, an ischemic condition in thekidney), a cardiovascular disease or disorder (e.g., hypertension,pulmonary hypertension, systolic hypertension, diabetic hypertension,asymptomatic left ventricular dysfunction, chronic congestive heartfailure, myocardial infarction, cardiac rhythm disturbance,atherosclerosis, etc.), cancer, heart disease or disorder, a kidneydisease or disorder, a gastrointestinal disease or disorder, a liverdisease or disorder, a lung disease or disorder, a pancreas disease ordisorder (e.g., pancreatitis), mental disease or disorder (e.g.,depression, anxiety, etc.), or a neurological disease or disorder. Insome embodiments, the method of diagnosing includes a further step oftreating the patient for the diagnosed disease or disorder.

In another embodiment, the invention is a method of assessing theseverity of a disease or disorder of a subject by assessing the level ofrenalase, or a renalase fragment, in a biological sample of the subject.In one embodiment, the biological sample of the subject is a cell,tissue, or bodily fluid. Non-limiting examples of bodily fluids in whichthe level of renalase, or a renalase fragment, can be assessed include,but are not limited to, blood, serum, plasma and urine. In variousembodiments, the level of renalase, or a renalase fragment, in thebiological sample of the subject is compared with the renalase, or arenalase fragment, level in a comparator. Non-limiting examples ofcomparators include, but are not limited to, a negative control, apositive control, an expected normal background value of the subject, ahistorical normal background value of the subject, an expected normalbackground value of a population that the subject is a member of, or ahistorical normal background value of a population that the subject is amember of. In various embodiments, the disease or disorder is acuterenal failure (i.e., acute tubular necrosis, or ATN, an ischemiccondition in the kidney), a cardiovascular disease or disorder (e.g.,hypertension, pulmonary hypertension, systolic hypertension, diabetichypertension, asymptomatic left ventricular dysfunction, chroniccongestive heart failure, myocardial infarction, cardiac rhythmdisturbance, atherosclerosis, etc.), cancer, heart disease or disorder,a kidney disease or disorder, a gastrointestinal disease or disorder, aliver disease or disorder, a lung disease or disorder, a pancreasdisease or disorder (e.g., pancreatitis), mental disease or disorder(e.g., depression, anxiety, etc.), or a neurological disease ordisorder. In some embodiments, the method of assessing the severityincludes a further step of treating the patient for the disease ordisorder.

In another embodiment, the invention is a method of monitoring theeffect of a treatment of a disease or disorder of a subject by assessingthe level of renalase, or a renalase fragment, in a biological sample ofthe subject. In one embodiment, the biological sample of the subject isa cell, tissue, or bodily fluid. Non-limiting examples of bodily fluidsin which the level of renalase, or a renalase fragment, can be assessedinclude, but are not limited to, blood, serum, plasma and urine. Invarious embodiments, the level of renalase, or a renalase fragment, inthe biological sample of the subject is compared with the renalase, or arenalase fragment, level in a comparator. Non-limiting examples ofcomparators include, but are not limited to, a negative control, apositive control, an expected normal background value of the subject, ahistorical normal background value of the subject, an expected normalbackground value of a population that the subject is a member of, or ahistorical normal background value of a population that the subject is amember of. In various embodiments, the disease or disorder is acuterenal failure (i.e., acute tubular necrosis, or ATN, an ischemiccondition in the kidney), a cardiovascular disease or disorder (e.g.,hypertension, pulmonary hypertension, systolic hypertension, diabetichypertension, asymptomatic left ventricular dysfunction, chroniccongestive heart failure, myocardial infarction, cardiac rhythmdisturbance, atherosclerosis, etc.), cancer, heart disease or disorder,a kidney disease or disorder, a gastrointestinal disease or disorder, aliver disease or disorder, a lung disease or disorder, a pancreasdisease or disorder (e.g., pancreatitis), mental disease or disorder(e.g., depression, anxiety, etc.), or a neurological disease ordisorder. In some embodiments, the method of monitoring the effect of atreatment includes a further step of treating the patient for thedisease or disorder.

In various embodiments, the subject is a human subject, and may be ofany race, sex and age. Representative subjects include those who aresuspected of having experienced a disease or disorder, those who havebeen diagnosed as having experienced a disease or disorder, those whohave been diagnosed as having a disease or disorder, and those who areat risk of developing a disease or disorder.

Information obtained from the methods of the invention described hereincan be used alone, or in combination with other information (e.g.,disease status, disease history, vital signs, blood chemistry, etc.)from the subject or from the biological sample obtained from thesubject.

In the diagnostic methods of the invention, a biological sample obtainedfrom a subject is assessed for the level of renalase, or a renalasefragment, contained therein. In one embodiment, the biological sample isa sample containing at least a fragment of a renalase polypeptide usefulin the methods described herein.

In other various embodiments of the methods of the invention, the levelof renalase is determined to be increased when the level of renalase, ora renalase fragment, is increased by at least 10%, by at least 20%, byat least 30%, by at least 40%, by at least 50%, by at least 60%, by atleast 70%, by at least 80%, by at least 90%, by at least 100%, by atleast 200%, by at least 300%, by at least 400%, by at least 500%, by atleast 600%, by at least 700%, by at least 800%, by at least 900%, by atleast 1000%, when compared to with a comparator control. In variousembodiments, an increased level of renalase, or a renalase fragment, isindicative of a disease or disorder. In various embodiments, the diseaseor disorder is acute renal failure (i.e., acute tubular necrosis, orATN, an ischemic condition in the kidney), cardiovascular disease, andcancer.

In the methods of the invention, a biological sample from a subject isassessed for the level of renalase, or a renalase fragment, in thebiological sample obtained from the patient. The level of renalase, or arenalase fragment, in the biological sample can be determined byassessing the amount of renalase polypeptide, or a fragment, in thebiological sample, the amount of renalase mRNA, or a fragment, in thebiological sample, the amount of renalase activity (e.g., enzymaticactivity, substrate binding activity, receptor binding activity, etc.)in the biological sample, or a combination thereof. In some embodiments,the level of renalase in the biological sample is determined in an assayusing at least one of the renalase binding molecules of the inventiondescribed elsewhere herein.

In various embodiments of the methods of the invention, methods ofmeasuring renalase levels in a biological sample obtained from a patientinclude, but are not limited to, an immunochromatography assay, animmunodot assay, a Luminex assay, an ELISA assay, an ELISPOT assay, aprotein microarray assay, a Western blot assay, a mass spectrophotometryassay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquidchromatography-tandem mass spectrometry assay, an ouchterlonyimmunodiffusion assay, reverse phase protein microarray, a rocketimmunoelectrophoresis assay, an immunohistostaining assay, animmunoprecipitation assay, a complement fixation assay, FACS, anenzyme-substrate binding assay, an enzymatic assay, an enzymatic assayemploying a detectable molecule, such as a chromophore, fluorophore, orradioactive substrate, a substrate binding assay employing such asubstrate, a substrate displacement assay employing such a substrate,and a protein chip assay (see also, 2007, Van Emon, Immunoassay andOther Bioanalytical Techniques, CRC Press; 2005, Wild, ImmunoassayHandbook, Gulf Professional Publishing; 1996, Diamandis andChristopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays inClinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, ProteomicsToday, John Wiley and Sons; 2007). In some embodiments, the level ofrenalase in the biological sample is measure with an assay that uses atleast one of the renalase binding molecules of the invention that aredescribed elsewhere herein.

Kits

The invention also includes a kit comprising a renalase binding molecule(e.g., antibody, etc.), or combinations thereof, of the invention and aninstructional material which describes, for instance, administering therenalase binding molecule, or a combination thereof, to an individual asa therapeutic treatment or a non-treatment use as described elsewhereherein. In an embodiment, this kit further comprises a (preferablysterile) pharmaceutically acceptable carrier suitable for dissolving orsuspending the therapeutic composition, comprising a renalase bindingmolecule, or combinations thereof, of the invention, for instance, priorto administering the renalase binding molecule of the invention to anindividual. Optionally, the kit comprises an applicator foradministering the renalase binding molecule.

Experimental Examples

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Development of Renalase Antibodies

Peptides were used as immunogens. The peptides generated ranged from 9to 21 amino acids and corresponded to regions of the renalase-1 andrenalase-2 proteins. All of the peptides had an N or C terminal cysteineresidue. The sequence of the peptides can be seen in Table 1 and wherethese peptides correspond to the renalase-1 or 2 sequences isdemonstrated in the sequence alignment of FIG. 4. As can be seen, therenalase-1 specific peptides are labeled 1A-F and the renalase-2specific peptide is labeled 3A5. Each peptide was conjugated to theadjuvant KLH via the cysteine and used to immunize 6 rabbits. Antiserumcollected from each animal was screened for anti-renalase antibody titerby ELISA assay using both the relevant peptide (BSA-conjugate) or fulllength renalase-1 or 2. The antisera were also tested for their abilityto detect endogenous renalase in tissue lysates by western blot. Usingthese screening criteria the animals producing antibodies with thepreferred characteristics were selected. In some examples and for somepeptides, several animals produced antibodies with the requiredspecificity. In these cases one animal had a final antisera bleed forpolyclonal antibody production and one or in some examples two otheranimals were used to harvest spleen lymphocytes. In other examples asingle animal had a terminal bleed and a splenectomy. Polyclonalantibodies raised against all of the peptides were generated bypurification of total IgG from terminal bleeds by protein Gchromatography followed by further purification on peptide affinitychromatography. Further and using standard procedures, lymphocytes fromthe spleens of selected animals were fused to myeloma cells forhybridoma generation. The hybridoma supernatants were screened forbinding to both the peptides against which they were raised andsecondarily screened against whole renalase protein. Selected hybridomaswere sub-cloned and expanded for antibody purification. The monoclonalantibodies were purified from conditioned hybridoma culture supernatantby protein A affinity chromatography.

TABLE 1 Sequence of renalase peptides used togenerate anti-renalase antibodies Mono- Antigen Antigen Speci- poly-Mono- clonal Code Sequence ficity clonal clonal Name 1A AVWDKADDSGGR1, R2 Yes RMTTAC 1B AVWDKAEDSGG R1, R2 Yes RMTTAC 1C CTPHYAKKHQR R1, R2Yes Yes 1C-22-1 FYDEL 1D CIRFVSIDNKK R1, R2 Yes Yes 1D-28-4 RNIESSEIGP1D-37-10 1E PGQMTLHHKPF R1, R2 Yes LAC 1F CVLEALKNYI R1 Yes Yes 1F-26-11F-42-7 3A PSAGVILGC R2 Yes Yes 3A-5-2Antibody Affinity determined by Biocore

Binding studies were performed using a Biacore T100. Binding studieswere performed at 25° C. using 25 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA,10% glycerol, 0.005% Tween-20 and 0.1 mg/mL BSA as the running buffer.The biotinylated antibodies were captured on individual streptavidinsensor chip flow cells as shown below. Because the study took two sensorchips, the analysis of two of the mAbs on the second sensor chip wasrepeated to gather more data. Renalase-1 was tested at 50 nM as thehighest concentration in a three-fold dilution series. Each of 5concentrations was tested in duplicate. Bound complexes were regeneratedwith a short pulse of 1/1000 phosphoric acid. Data sets were global fitto extract estimates of the binding constants summarized in Table 2.

TABLE 2 Affinity of renalase monoclonal antibodies as determined byBiocore IgG ka (M−1s−1) kd (s−1) KD (nM) BiO-1D 28-4 6.467(4)e42.04(3)e−5 0.316(5) BiO-1F 42-7 3.928(5)e4 9.47(6)e−5 2.41(2) BiO-1D37-10 1.749(5)e4 4.67(4)e−5 2.67(3) BiO-1F 26-1 4.526(8)e4 1.020(9)e−4 2.25(2) 1D28-4 has highest affinity and was used in the inhibitionstudies

The Nucleotide and Amino Acid Sequence of Anti-Renalase Antibodies

The monoclonal antibodies 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7 and 3A-5-2were selected for their renalase binding specificity and high affinity.Using standard polymerase chain reaction procedures and degenerateprimer sets, the cDNA of the antibody heavy and light chain variableregions for these antibodies were amplified from the subclonedhybridomas. The variable region nucleotide and amino acid sequences of1D-28-4 (RP-220) are shown in FIG. 5. In this way the composition ofantibodies with preferred characteristics is exemplified.

Inhibition of Renalase Signaling by Antibodies Decreases the Survival ofCancer Cells

Since renalase expression is up-regulated in several cancer cell lines(FIG. 6), experiments were performed to determine whether renalaseprovided a survival advantage to cancer cells. It was found thatrenalase expression increased markedly in nevi, and metastatic melanomacompared to normal skin (FIG. 7), suggesting that renalase provided asurvival advantage to melanocytes. In addition, RenMonoAb1 (monoclonalraised against RP-220) was highly effective at reducing the viability ofA375.S2 (melanoma cell line with mutant B-Raf (V600E)), and displayedsynergism with two alkylating agents active against melanoma:temozolomide, (FIG. 8) and dacarbazine (FIG. 9). RenMonoAb 1 was alsoeffective at reducing the viability of the melanoma cell line Sk-Mel-28(expresses mutant B-Raf (V600E) and wild type N-Ras) and showedsynergism with temozolomide (FIG. 10).

Next, experiments were performed to determine whether the inhibitoryaction of RenMonoAb 1 was specific to melanoma, or whether it affected abroader range of tumor cells. CCL-119 cells (CCF-MEC, acutelymphoblastic leukemic cell line; American Type Culture Collection)divide rapidly and express a high level of renalase, approximately3.8-fold over mean (microarray data from BioGPS.org) among the cellsmaking up the NCI-60 panel. RenMonoAb1 significantly decreased theviability of CCL-119 cells in culture (FIG. 11). Similarly, RenMonoAb1also inhibited the growth of two pancreatic cancer cell lines, MiaPacand Panc1 (FIGS. 12-13). FIG. 14 is a photomicrogarph depicting theeffect of renalase monoclonal antibody on melanoma cell number andmorphology. It was observed that renalase monoclonal (e.g., 1D-28-4)inhibits melanoma cells in culture. FIG. 15 shows that two additional,1C-22-1 and D-37-10 renalase monoclonal antibodies also inhibit melanomacell growth. These data indicate that renalase inhibition could be auseful therapeutic option in several cancers.

Renalase Overexpression Associated with Poor Outcome in MelanomaPatients

The expression of renalase in primary and metastatic tumor samplesobtained from Yale discovery and metastatic series (263 patientsfollowed for up to 30 years) was examined. Fluorescence-basedimmuno-histochemical staining was performed using the automatedquantitative analysis (AQUA) technology (Gould et al., 2009 Journal ofClinical Oncology, 27:5772-5780), a method by which target antigenexpression is determined within a compartment defined by labeling withboth anti S-100 and anti gp100. It was found that elevated renalaseexpression in melanoma tissue was associated with a significant increasein disease-specific mortality (FIG. 16), suggesting that inhibition ofrenalase's action may be a useful therapeutic option in this disease.

Example 2: Renalase Expression by Alternatively Activated, TumorAssociated Macrophages Promotes Melanoma Growth Through a STAT3 MediatedMechanism

Since RNLS functions as a survival factor that engages the MAPK and PI3Kpathways, and because its expression is regulated by STAT3 (Sonawane etal., 2014 Biochemistry. 53(44):6878-6892), the question is whether RNLSexpression and signaling provides a survival advantage to cancer cells.The focus is on melanoma, a disorder in which the MAPK, PI3K andJAK/STAT pathways are regulated abnormally, and for which additionaltherapeutic targets would be desirable.

RNLS expression is markedly increased in melanoma cell lines and tumorsamples. In patients with metastatic melanoma, RNLS expression isinversely correlated with disease-specific survival. Examination of thepattern of expression of RNLS in melanoma suggests that up-regulationpredominantly occurs in the cellular components of tumor-associatedstroma, specifically in CD163+ macrophages. Experimental data indicatethat alternatively activated macrophages (M2-like, CD163⁺) recruitedinto the tumors suppress the immune response against the tumor, increaseangiogenesis, and facilitate tumor cell migration, invasion anddissemination (Ruhrberg et al., 2010 Nat Med. 16:861-2; Pollard et al.,2004 Nat Rev Cancer. 4:71-8; Hao et al., 2012 Clinical and DevelopmentalImmunology. 2012:11). TAMs account for a significant percentage of thetumor mass in human melanoma, and also in the xenograft model describedin this work.

RNLS is preferentially expressed in CD162⁺ TAMs, suggesting that M2-likeTAMs could facilitate tumor progression by secreting RNLS. FIG. 22Cillustrates a working model that incorporates the key mechanisms thatunderlie the anti-tumor effects observed with inhibition of RNLSsignaling. Inhibition of RNLS signaling by the RNLS monoclonal m28-RNLSincreases the ratio of CD86⁺to CD163⁺ TAMs, and decreases RNLS secretionby CD163⁺ TAMs. In addition, m28-RNLS inhibits RNLS signaling inmelanoma cells. The net result is a dramatic fall in total andphosphorylated STAT3, leading to apoptosis.

The regulatory promoter elements and transcription factors that regulateRNLS gene expression have been recently investigated (Sonawane et al.,2014 Biochemistry. 53(44):6878-6892) and these data point to a key rolefor STAT3. The results suggest the existence of a feedforward loopbetween RNLS and STAT3, in which signals that upregulate STAT3 increaseRNLS gene expression, which in turn increases STAT3 activity. Theexistence of such an interaction between RNLS and STAT3 has importantimplications regarding the role of RNLS signaling in the pathogenesis ofcancer. Indeed, there are extensive data pointing to a key role for theSTAT family proteins, particularly STAT3, in the induction andmaintenance of an inflammatory microenvironment that facilitatesmalignant transformation and cancer progression (Yu et al., 2009 Nat RevCancer. 9:798-809). STAT3 signaling is often persistently activated inmalignant cells, and such activation not only drives tumor cellproliferation, but also increases the production of a large number ofgenes that sustain inflammation in the tumor microenvironment. A STAT3feedforward loop between cancer cells and non-transformed and stromalcells has been documented in cancer (Catlett-Falcone et al., 1999Immunity. 10:105-15; Yu et al., 2007 Nat Rev Immunol. 7:41-51; Ara etal., 2009 Cancer Res. 69:329-37). For instance, STAT3 is constitutivelyactivated in multiple myeloma patients. In the IL-6-dependent humanmyeloma cell line U266, IL-6 signals through Janus kinases to activateSTAT3, which in turn up-regulates anti-apoptotic factors, and promotesthe survival of tumor cells (Catlett-Falcone et al., 1999 Immunity.10:105-15). Through various mechanisms, STAT3 has also been found to beconstitutively activated in a majority of melanomas leading to increasesin tumor cell survival, proliferation, metastasis, angiogenesis, anddecreases in tumor immune response (Lesinski et al., 2013 Futureoncology. 9:925-7; Kortylewski et al., 2005 Cancer metastasis reviews.24:315-27; Emeagi et al., 2013 Gene therapy. 20:1085-92; Yang et al.,2010 International journal of interferon, cytokine and mediatorresearch: IJIM. 2010:1-7).

RNLS mediates cytoprotection by increasing the anti-apoptotic factorBcl2, and preventing the activation of effector caspases (Wang et al.,2014 Journal of the American Society of NephrologyDOI:10.1681/asn.2013060665). Inhibition of RNLS signaling in A375.S2cells is associated with sustained activation of p38 MAPK, followed byactivation of the apoptotic factor Bax, and apoptosis. The MAPK p38 is astress-activated protein kinase that has been implicated ininflammation, cell differentiation, cell cycle regulation and apoptosis(Ono et al., 2000 Cellular Signalling. 12:1-13). For instance, nervegrowth factor withdrawal was shown to cause apoptosis followingsustained activation of JNK and p38, and down-regulation of ERK (Xia etal., 1995 Science. 270:1326-31). However, since under certain conditionsinhibition of p38 can block apoptosis (Ono et al., 2000 CellularSignalling. 12:1-130, p38's role in apoptosis is clearly contextdependent. The data suggests that in A375.S2 cells, RNLS dependentactivation of p38 causes apoptosis.

Inhibition of RNLS signaling markedly decreases the expression of Ki-67in xenographs of melanoma. Since Ki-67 is a well-defined marker ofcellular proliferation that has been used extensively to evaluate theproliferative capacity of tumors, the data is interpreted as indicatingthat RNLS signaling is a key driver of tumor proliferation, and thatRNLS inhibition decreases the proliferative rate of tumors. Many of thekey factors that determine cell cycle progression have been identified,and include a set of cyclin dependent kinases (CDKs) along with twoclasses of CDK inhibitors, namely the inhibitor of cyclin dependentkinase 4 (INK4) and the CDK interacting protein/kinase inhibitor protein(CIP/KIP) families (Jung et al., 2010 Cellular Signalling. 22:1003-12).The expression of p21, a CDK inhibitor belonging to the CIP/KIP family,is regulated by RNLS signaling. Inhibition of RNLS signaling isassociated with a marked increase in p21 expression. p21 is a negativeregulator of cell cycle that can maintain cells in G0, block G1/Stransition, and cause G1 or inter-S phase arrest (Jung et al., 2010Cellular Signalling. 22:1003-12). Therefore, the increase in p21expression could account for the decrease in cell proliferation observedin tumors treated with an anti RNLS antibody. In addition, p38 has alsobeen shown to affect cell cycle progression (Ono et al., 2000 CellularSignalling. 12:1-13), and activation of p38 by anti RNLS treatment couldalso contribute to cell cycle arrest.

These findings identify RNLS as a secreted protein that can promote thesurvival and growth of cancer cells, and provide a framework to furtherinvestigate the use of anti RNLS therapy for the treatment of malignantmelanoma, alone or in conjunction with other TAM- or melanoma-inhibitingdrugs, such as CSF-1R inhibitors or MAPK pathway inhibitors,respectively. Because there are multiple mechanisms for regulating MAPKand PI3K and JAK/STAT3 and since there is crosstalk between pathways,cell fate depends on the dynamic balance and integration of multiplesignals, and the data suggests that RNLS inhibition will tilt thebalance toward cancer cell death.

The materials and methods used in this example are now described.

Reagents

Human melanoma cell lines A375.S2, SkMe128, SkMe15, MeWo, and WM266-4were obtained from the American Type Culture Collection and maintainedas recommended. Recombinant human RNLS was expressed, purified,concentrated, and dialyzed against PBS as described (Desir et al.,Journal of the American Heart Association. 2012; 1:e002634). RNLSpeptides RP220 and mutated peptide RP220A were synthesized at UnitedPeptide. Rabbit anti-RNLS monoclonal antibody (AB178700), goatpolyclonal anti-RNLS antibody (AB31291), goat IgG and rabbit IgG werepurchased from Abcam.

Synthesis of Anti-RNLS Monoclonal Antibodies m28-RNLS (Also Known as1D-28-4), m37-RNLS (also known as 1D-37-10)

RNLS peptide RP-220 was conjugated to KLH and used to immunize 6rabbits, and lymphocytes from the spleens of selected animals were fusedto myeloma cells for hybridoma generation. Hybridoma supernatants werescreened against rRNLS and selected hybridomas were cloned and expandedfor antibody purification. The monoclonal antibodies were purified fromconditioned hybridoma culture supernatant by protein A affinitychromatography.

Two clones, m28-RNLS (also known as 1D-28-4), m37-RNLS (also known as1D-37-10), were selected based on their high binding affinity (KD of0.316 and 2.67 nM, respectively) as determined using a Biacore T100system. The m28-RNLS' nucleotide sequence was determined by PCR,synthesized and cloned it into a mammalian expression vector. m28-RNLS,synthesized by transient expression into 293-F cells, was purified byprotein A chromatography.

Tissue Specimens

Human melanoma cDNA arrays I and II were obtained from OriGeneTechnologies (Rockville, Md., USA). The relevant pathology reports areavailable online: http://www.origene.com/assets/documents/TissueScan.Human melanoma and normal skin tissue samples obtained from US Biomax(Rockville, Md., USA) were used for immunohistochemistry orimmunofluorescence.

Quantitative RT-PCR

Relative expression levels of various genes were assessed by qRT-PCR, asdescribed previously (Lee et al., 2013 J Am Soc Nephrol. 24:445-55). ThemRNA level of RNLS, 2′-5′-oligoadenylate synthetase 1 (OAS1), β-actinand 18s rRNA was assessed using the TaqMan Gene Expression real-time PCRassays (Applied Biosystems, Carlsbad, Calif., USA). The results wereexpressed as the threshold cycle (Ct). The relative quantification ofthe target transcripts normalized to the endogenous control 18s rRNA orβ-actin was determined by the comparative Ct method (ΔCt) and the 2-ΔΔCtmethod was used to analyze the relative changes in gene expressionbetween the tested cell lines according to the manufacturer's protocol(User Bulletin No. 2, Applied Biosystems).

Immunohistochemical Staining and Western Blot Analysis

Immunohistochemistry was performed as described previously (Guo et al.,2012 Cancer science. 103:1474-80). Briefly, tumor tissues wereformalin-fixed, paraffin-embedded and cut into 5-μm sections on glassslides. The slides were de-paraffinized and hydrated, followed byantigen retrieval in a pressure cooker containing 10 mM sodium citrate,pH6 buffer. The sections were blocked in 3% hydrogen peroxide for 30 minand 2.5% normal horse serum in PBS/0.1% Tween20 for 1 h followed byincubation with primary antibody and isotype control IgG overnight at 4°C. The following antibodies were used in this study: m28-RNLS at 500ng/ml; goat polyclonal anti-RNLS at 250 ng/ml (Abcam, ab31291); rabbitmonoclonal anti-CD68 (BDBioscience, 1:100); rabbit monoclonal anti-CD163(AbD Serotec, 1:100); rabbit monoclonal anti-CD86 (Abcam, 1:100); rabbitmonoclonal anti-Ki67 (Vector Lab, VP-RM04, 1:100); rabbit monoclonalanti-p21, phspho-Tyr⁷⁰⁵-Stat3, and total Stat3 (Cell SignalingTechnologies, #2947, 1:100, #9145, 1:400, and #4904, 1:400,respectively). ImmPRESS peroxidase-anti-rabbit IgG (Vector Laboratories,Burlingame, Calif., USA) was used to detect primary antibodies. Thecolor was developed using a Vector DAB substrate kit and counterstainedwith hematoxylin (Vector Laboratories). Slides were observed andphotographed using an Olympus BX41 microscope and camera (OlympusAmerica Inc, Center Valley, P A, USA).

Western blot analysis was carried out as previously described (Wang etal., 2014 Journal of the American Society of NephrologyDOI:10.1681/asn.2013060665).

Tissue Microarray

Melanoma tissue microarrays were purchased from US BioMax, Inc. and Yaletissue pathology services. This study was approved by the HumanInvestigation Committee of Yale University School of Medicine (HICprotocol No. 1003006479). The Yale melanoma tissue microarray wasconstructed as previously described (Berger et al., 2003 Cancerresearch. 63:8103-7; Rimm et al., 2001 Cancer journal. 7:24-31). A totalof 570 tissue cores representing 542 total melanoma cases and a smallseries of controls measuring 0.6 mm were spaced 0.8 mm apart on a singleglass slide. The cohort was constructed from formalin-fixed,paraffin-embedded tissue blocks obtained from the archives of theDepartment of Pathology at Yale University School of Medicine. Apathologist examined each case to select the region for inclusion in thetissue microarray. Core biopsies from the specimens were placed on thetissue microarray with a Tissue Micorarrayer (Beecher Instruments, SunPrairie, Wis.). The tissue microarrays were then cut to 5-um sectionsand placed on glass slides with the adhesive tape transfer system(Instumedics, Inc., Hackensack, N.J.) with UV cross-linking. Thespecimens were all drawn from archives of tumors resected between 1959and 1994, with a follow-up range of 2 months and 38 years (medianfollow-up time, 60 months). The cohort characteristics are describedpreviously (Berger et al., 2004 Cancer research. 64:8767-72).

The tissue microarray slide was stained as described previously (Bergeret al., 2004 Cancer research. 64:8767-72; Nicholson et al., 2014 Journalof the American College of Surgeons. 219:977-87). The slides weredeparaffinized, rehydrated, unmasked, and blocked in the same way asprocessed for immunohistochemistry described above. The melanoma tissuearrays were stained with a cocktail of m28-RNLS plus anti-S100 mousemonoclonal (1:100, Millipore, Temecula, Calif., USA) and anti-HMB45mouse monoclonal (1:100, Thermo Scientific, Fremont, Calif., USA)diluted in BSA/TBS at 4° C. overnight. The secondary antibodies Alexa488-conjugated goat anti-mouse (1:100, Molecular Probes, Eugene, Oreg.)plus Envision anti-rabbit (DAKO) diluted in BSA/TBS were applied for 1hour at room temperature. The slide was washed with TBST (three timesfor 5 minutes each) and then incubated with Cy5-tyramide (Perkin-ElmerLife Science Products, Boston, Mass.) and activated by horseradishperoxidase, resulting in the deposition of numerous covalentlyassociated Cy5 dyes immediately adjacent to the horseradishperoxidase-conjugated secondary antibody. Cy5 was used because itsemission peak (red) is well outside of the green-orange spectrum oftissue autofluorescence. The slides were sealed with coverslips withProlong Gold anti-fade reagent containing 4′,6-Diamidino-2-phenylindoleto visualize nuclei.

Cell Viability Assays

Total cell number and percentage of live cells were assessed by trypanblue exclusion, and cells were counted using a BioRad TC10 automatedcell counter. For additional studies, cell viability was determinedusing WST-1 reagent (Roche Diagnostics, Indianapolis, Ind., USA)according to the manufacturers' instruction. Absorbances were read usinga microplate reader (Power Waves XS, BioTek Instruments, Winooski, Vt.,USA).

RNA Interference

Four individual siRNAs and a siRNA SMART pool targeting RNLS werepurchased from Dharmacon (Lafayette, Colo., USA). Cells were transfectedwith RNLS siRNA or a universal negative control small interfering RNA(control siRNA, Dharmacon) using DharmaFECT 4 reagent (Dharmacon) asinstructed by the manufacturer. Knock-down efficiency was determined byqPCR.

Mouse Tumor Model

Female athymic, 18-20 g nude mice (nu/nu) were obtained from CharlesRiver (Willimantic, Conn.) and housed in microisolator cages, withautoclaved bedding in a specific pathogen-free facility, with a 12-hlight/dark cycle. Animals received water and food ad libitum, and wereobserved for signs of tumor growth, activity, feeding and pain, inaccordance with the study protocol approved by the VACHS IACUC.

Xenograft tumors were established by subcutaneous injection of A375.S2cells (2×10⁶ in 100 μl of PBS, pH 7.6). When the tumors reached a volumeof 50-100 mm³, the mice were divided into a control group (n=14 treatedwith rabbit IgG, 40 μg by intraperitoneal injection (IP) once weekly,and 40 ug subcutaneously (SQ) around the tumor site every 3 days), andan experimental group (n=14) that received m28-RNLS (40 μg IP, onceweekly, and 40 ug SQ, every 3 days). Tumor size was measured withdigital calipers and volume was calculated according to the formula(length× width)×π/2.

At the end of the study, the mice were sacrificed, the tumors wereexcised and immediately snap-frozen in liquid nitrogen and stored at−80° C. Apoptosis was examined using the TUNEL assay (Roche in situApoptosis Detection System), according to the manufacturer'sinstructions. Sections were examined by light microscopy and theapoptosis index was determined by counting ≧1000 cells in 10 randomlyselected high-power fields (×200 magnification).

Statistical Analyses

The Wilcoxon rank sum test and the Mann-Whitney U test were used forpaired and unpaired data, respectively. When appropriate fornonparametric repeated-measures, ANOVA (Friedman test) was used toevaluate statistical significance. When the Friedman test revealedstatistical significance, Dunn's test was used for pairwise comparisons.A Kaplan-Meier survival analysis and multivariate Cox regressionanalysis were also carried out. All data are mean±standard error of themean (mean±SEM), and values of P<0.05 were accepted as a statisticallysignificant difference. Statistical analyses of tissue array data wereperformed using SPSS® software, version 21.0 (SPSS Inc., Chicago, Ill.,USA).

The results of this example are now described.

RNLS Overexpression in Melanoma

In order to determine if RNLS expression differed between normal humanskin and malignant melanoma, tissue microarrays (TMAs; Yale TissueMicroarray Facility and US Biomax, Inc.) spanning the progression ofnormal skin to benign nevus to primary and metastatic melanoma wereexamined. The Yale TMAs contained formalin-fixed, paraffin-embeddedspecimens obtained from a cohort of 192 primary melanomas collectedduring 1959 to 1994, a cohort of 246 serial primary and metastaticmelanomas collected from 1997 to 2004, a cohort of 295 patients withbenign nevi, and matched normal skin specimens from 15 patients. Thedemographics and clinical characteristics for these tissue microarrayshave been described previously (Gould Rothberg et al., 2009 Journal ofclinical oncology: official journal of the American Society of ClinicalOncology. 27:5772-80). The US Biomax array contained 74 specimens,including 35 primary melanomas, 11 metastatic lesions, 14 benign nevi,and 14 normal samples. Examination of approximately 600 histospots forRNLS protein expression using a quantitative, automatedimmunofluorescence (IF) microscopy system (AQUA), revealed thatprogression from normal skin to benign nevi to primary malignantmelanoma to metastatic melanoma was accompanied by a significantincrease in RNLS expression (p=0.009, p=0.0003, and p<0.001,respectively, FIG. 17A-C).

The question is whether dysregulated RNLS expression and signaling couldfacilitate melanoma growth, and, therefore, serve as a prognosticmarker. Each primary melanoma from a cohort of 246 serial primary andmetastatic samples collected from 1997 to 2004 were examined. Onehundred nineteen patients had histospots that were suitable forevaluation by AQUA technology. In this group, the outcome of patientswhose tumors expressed high RNLS levels (RNLS AQUA score>median AQUAscore 75,764.45) were compared to those with low RNLS expression. HighRNLS expression was associated with increased melanoma-specific death:5-year and 10-year disease-specific survival rates of 55% versus 69% and39.7% versus 58.5%, respectively, p=0.008, (FIG. 17D). Followingmultivariate analysis of this cohort, RNLS levels were found to beindependently predictive of survival in melanoma (p=0.004, HR=3.130).Stage of disease at diagnosis (p=0.05, HR=3.940), Clark level (p=0.015,HR=1.687), and ulceration of the primary tumor (p=0.001, HR=2.54) werealso found to independently predict survival in melanoma. These findingssuggest that RNLS expression may serve as a useful prognostic marker inmelanoma, and may help identify a subset of patients with a moreaggressive phenotype.

RNLS Overexpression Favors Cancer Cell Survival

RNLS-mediated signaling is anti-apoptotic, and protects normal cellsexposed to toxic stress from apoptotic death (Wang et al., 2014 J Am SocNephrol.; Lee et al., 2013 J Am Soc Nephrol. 24:445-55). To explore ifRNLS signaling favored the survival of cancer cells, either recombinantRNLS (rRNLS) or bovine serum albumin (BSA) was added to serum-starvedmelanoma cells (A375.S2, MeWo, SkMe15, and SkMe128) in culture, and cellviability was determined. Compared to BSA, RNLS markedly increased thesurvival of serum-starved cells, and caused an apparent increase in theproliferative rate as measured by the WST-1 assay (n=6, p<0.05, FIG.18A). The total cell number and percentage of live cells of thosetreated with RNLS were counted to determine if the apparent increase inproliferative rate was due an increase in cell proliferation or to adecrease in the rate of apoptosis. As shown in FIG. 18B, treatment withRNLS showed increased cell counts, and increased percentage of livecells compared to those treated with BSA, suggesting that RNLS functionsas an anti-apoptotic, survival factor.

Inhibition of RNLS Signaling is Cytotoxic to Melanoma Cells In Vitro

Three approaches to determine the functional consequences of inhibitingRNLS expression and signaling in melanoma were used. First, the effectof decreasing RNLS expression on cell viability was evaluated. RNLSknockdown by siRNA markedly reduced the viability of the melanoma celllines A375.S2 and SkMe128 (p=0.03 and p=0.003, respectively, FIG. 19A).Second, since the RNLS peptide RP-220 mimics the protective effect andsignaling properties of rRNLS, it has been reasoned that it likelyinteracts with a critical region of the receptor for extracellular RNLSand that antibodies directed against it could have inhibitoryproperties. Therefore, a panel of monoclonal antibodies against RP-220was developed, and their effect on cancer cell survival was tested. Twomonoclonal antibodies generated against RNLS, [clones #28-4 (m28-RNLS),37-10 (m37-RNLS)] decreased the viability of all (total of 5) melanomacell lines tested, and representative examples are shown in FIGS. 19B-C.m28-RNLS demonstrated increasing levels of cytotoxicity in correlationwith increasing treatment concentrations (p<0.05, FIG. 19B). Third, apeptide antagonist (RP-220A) was generated by decreasing RP-220's netcharge (3 Lysine/arginine changed to alanine FIG. 19D). RP220A does notmediate RNLS dependent signaling, but binds to PMCA4b and antagonizesthe action of endogenous RNLS (Wang et al., 2015 PLoS ONE. 10:e0122932).RP-220A proved to be cytotoxic in increasing doses to melanoma cells inculture (p<0.005, FIG. 19D).

Inhibition of RNLS Signaling Blocks Tumor Growth In Vivo

A375.S2 (human melanoma) cells were injected subcutaneously into athymicnude mice to generate tumors. Once the tumors reached a volume of ˜50mm³, the animals were then treated with either control rabbit IgG or aRNLS neutralizing monoclonal antibody, m28-RNLS. As overall animalhealth and activity was maintained throughout the study, the antibodytreatment did not appear to be toxic. Tumor size was measured everyother day, and treatment with m28-RNLS decreased tumor volume at allpoints tested (p<0.05, FIG. 20A). The animals were sacrificed at day 11due to overall tumor size and ulceration in some animals. IHC stainingof sections from the xenografted tumors with the cellular proliferationmarker Ki67 revealed a significant decrease in cellular proliferationwithin the tumors treated with the anti-RNLS antibody versus to thosetreated with rabbit IgG: of 35.1±2.3 positive cells/high power field inthe control group vs. 13.4±3.0 in the RNLS Ab treated group, n=14,p=0.0004 (FIG. 20B).

Inhibition of RNLS Signaling Blocks Endogenous RNLS Expression and STAT3Activation and Induces Apoptosis and Cell Cycle Arrest

STAT3 is known to bind to the promoter region of the RNLS gene andincrease its expression, and a positive RNLS-STAT3 feedback loop hasbeen suggested (Sonawane et al., 2014 Biochemistry. 53(44):6878-6892).This relationship was further investigated through immunofluorescenttissue staining and study of the cell lysates from the xenograftedtumors treated with control IgG and m28-RNLS. Significant coexpressionof RNLS with phosphorylated and total STAT3 was noted in the tumorsamples (FIG. 21A) as assessed by IF. Treatment with m28-RNLS caused adramatic reduction in RNLS protein expression, and in both total andphosphorylated STAT3 (FIG. 21A). Changes in protein expression wereconfirmed by western blot as shown in FIGS. 21B-C. In tumors treatedwith m28-RNLS, STAT3 phosphorylation at tyrosine 705 (p-Y⁷⁰⁵-STAT3), andtotal STAT3 were significantly decreased (n=8, p<0.005, FIG. 21B-C).

To test if the significant decrease in RNLS expression was primarilyoccurring in the melanoma cells, human and mouse specific primers wereused to amplify tumor (human) and endogenous (mouse) RNLS in the tumormass. As depicted in FIG. 21D, treatment with m28-RNLS causes asignificant reduction in mouse RNLS expression, without affecting human(tumor) expression, suggesting that tumor-infiltrating cells play a keyrole in RNLS production and secretion.

In addition, increased expression of the cell cycle inhibitor p21 wasnoted. Antibody treatment markedly increased the expression of the cellcycle regulator p21 in the tumor samples: 24.2±2.4 positive cells in theantibody treated group vs. 12.2±1.0 in the control group, n=14, p=0.009(FIG. 21E). Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining revealed a significant increase in the average numberof cells undergoing apoptosis in the antibody-treated tumors over thecontrol group with an average of 13.3±0.6 positive cells vs. 4.3±0.2,n=14, p<0.001, (FIG. 21E). The increase in apoptosis was temporallyrelated to phosphorylation of p38 MAPK, and subsequent activation of theB-cell lymphoma 2 related protein Bax (FIG. 21F). These data indicatethat treatment with anti-RNLS antibody causes a marked reduction intotal and phosphorylated STAT3, decreases cell proliferation, andincreases apoptosis in tumor cells.

Inhibition of RNLS Signaling Increases the Ratio of CD86⁺ to CD163+ TAMs

The melanocytes did not appear to be the main source of the RNLS in themelanoma histospots, as there was minimal overlap noted between RNLS andmelanocyte staining (FIG. 17A). Melanomas often have significantinfiltration of immune cells, including macrophages. The infiltratingmacrophages appeared to contribute the majority of the tumoral RNLS as asubstantial component of the RNLS staining noted in each histospotoverlapped significantly with the pan-macrophage marker CD68 (FIG. 22Atop panel). Upon further investigation, it was determined that RNLS wascoexpressed predominantly with CD163+(M2-like) TAMs (FIG. 22A, middlepanel). Coexpression of RNLS with CD86+(M1-like) macrophages was minimal(FIG. 22A, bottom panel). M2-like (CD163+) macrophages are associatedwith immune escape and shown to promote cancer development and spread,while M1-like (CD86+) macrophages are typically pro-inflammatory, andinhibit tumor growth (Biswas et al., 2010 Nat Immunol. 11:889-96;Mantovani et al., Trends in Immunology. 23:549-55). Treatment of thexenografts with m28-RNLS antibody led to a considerable decrease in thenumber of CD163+ TMAs, and the remaining cells did not expressdetectable levels of RNLS (FIG. 22B).

Example 3: Sustained Renalase Signaling Through the Plasma MembraneCalcium ATPase PMCA4b Promotes Pancreatic Cancer Growth

Since RNLS functions as a survival factor that engages the MAPK and PI3Kpathways that are disordered in pancreatic cancer, and because itsexpression is regulated by the signal transducer and activator oftranscription STAT3 (Sonawane et al., 2014 Biochemistry.53(44):6878-6892), it has been postulated that abnormal regulation ofRNLS expression and signaling could provide a survival advantage tocancer cells, and promote tumor formation (Guo et al., 2014 Curr OpinNephrol Hypertens. 23(5):513-8).

It is shown herein that RNLS expression is increased in several types ofcancers, and in a cohort of patients with pancreatic ductaladenocarcinoma (PDAC), overall survival was inversely correlated withRNLS expression in the tumor, suggesting a pathogenic role for RNLS.Inhibition of RNLS expression using siRNA, or inhibitory anti-RNLSantibodies decreased cultured PDAC cells viability. In a xenograft mousemodel, the RNLS monoclonal antibody m28-RNLS inhibited PDAC growth, andcaused apoptosis and cell cycle arrest by down-regulating STAT3, andup-regulating in p21, and p38. Down-regulation of RNLS expression intumor cells led to an equivalent decrease in PMCA4b (RNLS receptor)expression and resulted in a reduction in tumor size similar to thatobserved with inhibitory anti-RNLS antibodies. These results reveal apreviously unrecognized pro-survival function of the RNLS pathway incancer, show that RNLS expression may serve as a prognostic marker, andidentify novel therapeutic targets for the management of pancreaticcancer.

Evidence is provided here for both a pathogenic role of increased RNLSexpression in PDAC, and for the therapeutic utility of inhibiting RNLSsignaling. In addition, the molecular mechanisms that mediate theobserved antitumor activity of inhibitors of RNLS signaling are beingexplored.

Taken together, these findings indicate that upregulated RNLS-mediatedsignaling plays a pathogenic role in PDAC. It is being shown here thathigh RNLS tumor expression is associated with a two-fold increase inoverall 3-year mortality, supporting the use of RNLS as a diagnostic orprognostic marker. Furthermore, since RNLS is a secreted protein, it canbe used as a biomarker for the primary detection of tumors, or as asurrogate marker for treatment response or recurrence.

A primary mechanism of RNLS mediated cyto-protection appears to be itsability to activate AKT, ERK and STAT, to increase the anti-apoptoticfactor Bcl2, and to prevent the activation of effector caspases (Wang etal., 2014 Journal of the American Society of Nephrology.DOI:10.1681/asn.2013060665). Inhibition of RNLS signaling in Panc1 cellsis associated with sustained activation of p38 MAPK, and apoptosis. p38is a stress-activated kinase that has been implicated in inflammation,cell differentiation, cell cycle regulation and apoptosis (Ono et al.,2000 Cellular Signalling. 12(1):1-13). For example, nerve growth factorwithdrawal causes apoptosis along with sustained activation of JNK andp38, and down-regulation of ERK (Xia et al., 1995 Science.270(5240):1326-31). However, since under certain conditions inhibitionof p38 can block apoptosis (Ono et al., 2000 Cellular Signalling.12(1):1-13), p38's role in the apoptotic process is clearly contextdependent. The data described herein are consistent with the explanationthat in Panc1 cells, m28-RNLS dependent activation of p38 is associatedwith apoptosis.

Inhibition of RNLS signaling markedly decreases the expression of Ki-67in xenografts of pancreatic cancer. Since Ki-67 is used to evaluatelevels of cell division, the data is consistent with the explanationthat RNLS inhibition decreases the proliferative rate of tumors. Many ofthe key factors that determine cell cycle progression have beenidentified, and include cyclin dependent kinases (CDK) and two classesof endogenous CKD inhibitors, namely the inhibitor of cyclin dependentkinase 4 (INK4) and the CDK interacting proteins/kinase inhibitor(CIP/KIP) protein families (Jung et al., 2010 Cellular Signalling.22(7):1003-12). The data reveal that the expression of p21, a CKDinhibitor belonging to the CIP/KIP family, is regulated by RNLSsignaling. Inhibition of RNLS signaling is associated with a markedincrease in p21 expression. Since p21 is a negative regulator of cellcycle that can maintain cells in G0, block G1/S transition and cause G1or inter-s phase arrest (Jung et al., 2010 Cellular Signalling.22(7):1003-12), its upregulation could account for the decrease in cellproliferation observed in tumors treated with m28-RNLS. In addition, p38has also been shown to affect cell cycle progression (Ono et al., 2000Cellular Signalling. 12(1):1-13), and its activation by anti-RNLStreatment could also contribute to cell cycle arrest.

The regulatory promoter elements and transcription factors that regulateRNLS gene expression have been recently investigated (Sonawane et al.,2014 Biochemistry. 53(44):6878-6892), and these data point to a key rolefor STAT3. The results suggest a feed-forward loop between RNLS andSTAT3: signals that upregulate STAT3 increase RNLS gene expression, andRNLS, in turn, increases STAT3 activity. Such an interaction betweenRNLS and STAT3 has important implications regarding the role of RNLSsignaling in the pathogenesis of cancer. STAT family proteins,particularly STAT3, are firmly implicated in the induction andmaintenance of an inflammatory microenvironment that facilitatesmalignant transformation and cancer progression (Yu et al., 2009 Nat RevCancer. 9(11):798-809). STAT3 signaling is often persistently activatedin cancer cells, and such activation not only drives tumor cellproliferation, but also increases the production of a large number ofgenes that sustain inflammation in the tumor microenvironment. A STAT3feed-forward loop between cancer cells and non-transformed and stromalcells has been documented in cancer (Catlett-Falcone et al., 1999Immunity. 10(1):105-15; Yu et al., 2007 Nat Rev Immunol. 7(1):41-51; Araet al., 2009 Cancer Res. 69(1):329-37). For instance, STAT3 isconstitutively activated in multiple myeloma patients. In theIL-6-dependent human myeloma cell line U266, IL-6 signals through Januskinases to the activate STAT3, which in turn up-regulates anti-apoptoticfactors, and promotes the survival of tumor cells (Catlett-Falcone etal., 1999 Immunity. 10(1):105-15). Likewise, STAT3 is constitutivelyactivated in the majority of pancreatic ductal adenocarcinomas, andappears to be required for the initiation and progression ofKRAS-induced pancreatic tumorigenesis (Corcoran et al., 2011 Cancer Res.71(14):5020-9).

The STAT3 pathway and RNLS may also have a role in promoting the mostcommon and important environmental factor in PDAC development, cigarettesmoking (Muscat et al., 1997 Cancer epidemiology, biomarkers &prevention: a publication of the American Association for CancerResearch, cosponsored by the American Society of Preventive Oncology.6(1):15-9; Boyle et al., 1996 International journal of cancer Journalinternational du cancer. 67(1):63-71; Fuchs et al., 1996 Archives ofinternal medicine. 156(19):2255-60). Nicotine, a key constituent ofcigarette smoke, has been shown to enhance the rate of proliferation andangiogenesis in cancers (Heeschen et al., 2002 J Clin Invest.110(4):527-36; Heeschen et al., 2001 Nat Med. 7(7):833-9). Nicotine'saction of tumor growth and metastases is believed to be mediated by itsinteraction with acetylcholine receptor alpha-7nACHR resulting inJAK-STAT3 and MEK-ERK1-2 downstream signaling cascades (Momi et al.,2013 Oncogene. 32(11):1384-95). In this context, nicotine increases RNLSpromoter activity through the synergistic action of Sp1 and STAT3(Sonawane et al., 2014 Biochemistry. 53(44):6878-6892).

PMCA4b has previously been characterized as a plasma membrane ATPaseinvolved in cell signaling, cardiac hypertrophy, and cancer (Cartwrightet al., 2007 Annals of the New York Academy of Sciences. 1099(1):247-53;Pinton et al., 2001 EMBO J. 20(11): 2690-2701; Oceandy et al., 2011Biochimica et Biophysica Acta (BBA)—Molecular Cell Research.1813(5):974-8). It transports Ca²⁺ from the cytosol to the externalenvironment, and appears to regulate local calcium concentration. Inaddition to its role in regulating cytoplasmic Ca²⁺, PMCA4b is centralto a macromolecular complex that can also signal through Ras and theMAPKs (Ara et al., 2009 Cancer Res. 69(1):329-37; Corcoran et al., 2011Cancer Res. 71(14):5020-9; Muscat et al., 1997 Cancer epidemiology,biomarkers & prevention: a publication of the American Association forCancer Research, cosponsored by the American Society of PreventiveOncology. 6(1):15-9). For example, it modulates Ras signaling and ERKactivation through its interaction with the tumor suppressor RASSF1(Armesilla et al., 2004 Journal of Biological Chemistry.279(30):31318-28). The data indicate that RNLS signals though PMCA4b,that down-regulation of PMCA4b expression or inhibition of its enzymaticfunction is cytotoxic to pancreatic adenocarcinoma cells. These findingssuggest that PMCA4b represent a therapeutic target in the management ofPDAC.

In summary, these findings demonstrate that RNLS is a secreted proteinthat can promote the survival and growth of PDACs. This provides aframework to further investigate the use of therapies that inhibit RNLSfor the treatment of cancer. In this context, that RNLS modulates themultiple inter-related signals that mediate MAPK, PI3K and JAK-STAT3 areactive in cancer, the molecule might be a particularly attractivetherapeutic target (FIG. 27E).

The materials and methods used in this example are now described.

Reagents

The human ductal pancreatic adenocarcinoma cell lines BxPC-3, Panc1 andMiaPaCa-2 were obtained from the American Type Culture Collection (ATCC)(Manassas, Va., USA) and maintained as recommended. The p38 and STAT3blockers SB203580 and Stattic were purchased from Abcam (Cambridge, UK).The JNK inhibitor SP600125 and the ERK inhibitor U0126 were obtainedfrom Sigma Aldrich (St. Louis, Mo., USA), and Cell SignalingTechnologies (Beverly Mass., USA), respectively. Recombinant human RNLS(rRNLS) was expressed, purified, concentrated, and dialyzed against PBSas previously described (Desir et al., 2012 J Am Heart Assoc.1(4):e002634). Rabbit anti-RNLS monoclonal (AB178700), goat polyclonalanti-RNLS (AB31291), goat IgG and rabbit IgG were purchased from Abcam.

Synthesis of Anti-RNLS Monoclonal Antibodies m28-RNLS (Also Known as1D-28-4), m37-RNLS (Also Known as 1D-37-10)

RNLS peptide RP-220 was conjugated to KLH and used to immunize 6rabbits, and lymphocytes from the spleens of selected animals were fusedto myeloma cells for hybridoma generation. Hybridoma supernatants werescreened against rRNLS and selected hybridomas were cloned and expandedfor antibody purification. The monoclonal antibodies were purified fromconditioned hybridoma culture supernatant by protein A affinitychromatography.

Two clones, m28-RNLS, m37-RNLS, were selected based on their highbinding affinity (KD of 0.316 and 2.67 nM respectively) as determinedusing a Biacore T100 system. The m28-RNLS' nucleotide sequence wasdetermined by PCR, synthesized and cloned it into a mammalian expressionvector. m28-RNLS, synthesized by transient expression into 293-F cells,was purified by protein A chromatography.

Tissue Specimens

Human cancer cDNA arrays (Screen cDNA Arrays I and II, pancreatic cancercDNA array) were obtained from OriGene Technologies (Rockville, Md.,USA). The relevant pathology reports are available online:www.origene.com/assets/documents/TissueScan. Human pancreas cancer andnormal tissue samples obtained from US Biomax (Rockville, Md., USA) wereused for immunohistochemistry or immunofluorescence.

Quantitative PCR

Relative expression levels of various genes were assessed by qPCR. ThemRNA level of RNLS, 2′-5′-oligoadenylate synthetase 1 (OAS1), β-actinand 18s rRNA was assessed using the TaqMan Gene Expression real-time PCRassays (Applied Biosystems, Carlsbad, Calif., USA). The results wereexpressed as the threshold cycle (Ct). The relative quantification ofthe target transcripts normalized to the endogenous control 18s rRNA orβ-actin was determined by the comparative Ct method (ΔCt) and the 2-ΔΔCtmethod was used to analyze the relative changes in gene expressionbetween the tested cell lines according to the manufacturer's protocol(User Bulletin No. 2, Applied Biosystems).

Immunohistochemistry and Western Blot Analysis

Immunohistochemistry was performed as described previously (Guo et al.,2012 Cancer Science. 103(8):1474-80). Briefly tumor tissues wereformalin-fixed, paraffin-embedded and cut into 5 μm sections on glassslides. The slides were de-paraffinized and hydrated, followed byantigen retrieval in a pressure cooker containing 10 mM sodium citrate,pH6 buffer. The sections were blocked in 3% hydrogen peroxide for 30 minand 2.5% normal horse serum in PBS/0.1% Tween20 for 1 h followed byincubation with primary antibody and isotype control IgG overnight at 4°C. The following antibodies were used in this study: m28-RNLS at 500ng/ml; goat polyclonal anti-RNLS at 250 ng/ml (Abcam, AB31291); rabbitmonoclonal anti Ki67 (Vector Lab, VP-RM04, 1:100); rabbit monoclonalanti p21 and phspho-Tyr⁷⁰⁵-Stat3 (Cell Signaling Technologies, #2947,1:100 and #9145, 1:400, respectively). ImmPRESS peroxidase-anti-rabbitIgG (Vector Laboratories, Burlingame, Calif., USA) was used to detectprimary antibodies. The color was developed using a Vector DAB substratekit and counterstained with hematoxylin (Vector Laboratories). Slideswere observed and photographed using an Olympus BX41 microscope andcamera (Olympus America Inc, Center Valley, Pa., USA).

Western blot analysis was carried out as previously described (Wang etal., 2014 Journal of the American Society of Nephrology. DOI:10.1681/asn.2013060665).

Tissue Microarray

Pancreas tissue microarrays were purchased from US BioMax. Tissuemicroarray slides were stained as described previously (Nicholson etal., 2014 Journal of the American College of Surgeons. 219(5):977-87).In brief, specimens were co-stained with m28-RNLS and mouse monoclonalpan-cytokeratin antibodies (1:100, DAKO M3515) at 4° C. overnight. Thesecondary antibodies Alexa 488-conjugated goat anti-mouse (1:100,Molecular Probes, Eugene, Oreg.) and Envision anti-rabbit (DAKO) wereapplied for 1 hour at room temperature. The slides were washed withTris-buffered saline (three times for 5 minutes), and incubated withCy5-tyramide (Perkin-Elmer Life Science Products, Boston, Mass.) andactivated by horseradish peroxidase. Cy5 was used because its emissionpeak (red) is outside of the green-orange spectrum of tissueauto-fluorescence. The slides were sealed with coverslips with ProlongGold anti-fade reagent containing 4′,6-Diamidino-2-phenylindole tofacilitate the visualization of nuclei.

Cell Viability Assays

Cell viability was assessed by trypan blue exclusion, and cells werecounted using a BioRad TC10 automated counter. For some studies, cellviability was determined using the WST-1 reagent (Roche Diagnostics,Indianapolis, Ind., USA) as previously described (Wang et al., 2014Journal of the American Society of Nephrology.DOI:10.1681/asn.2013060665).

Apoptosis and Cell Cycle Analysis

For cell cycle analysis, cultured cells were dissociated using 10 mMEDTA, fixed with ice-cold 70% ethanol, digested with RNAse A, andstained with propidium iodide. Propidium staining was detected using aBD FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA),and analyzed using CellQuest software.

Apoptosis was detected and quantified as previously done (Guo et al.,2012 Cancer Science. 103(8):1474-80). In brief, cells were stained withFITC-labeled Annexin-V and propidium iodide according to themanufacturer's instructions (Bender MedSystems, Burlingame, Calif.,USA). At least 20,000 events were collected on a BD FACSCalibur flowcytometer (BD Biosciences, San Jose, Calif., USA) and analyzed usingCellQuest software.

RNA Interference

Four individual siRNAs and a siRNA SMART pool targeting RNLS werepurchased from Dharmacon (Lafayette, Colo., USA). Cells were transfectedwith RNLS siRNA or a universal negative control siRNA (control siRNA,Dharmacon) using DharmaFECT 4 reagent (Dharmacon) as suggested by themanufacturer.

To generate a stably transfected Panc1 cell line, cells were transducedwith lentivirus (Santa Cruz) carrying either RNLS shRNA (sh-RNLS) orcontrol shRNA (sh-Control) according to the manufacturer's protocol.Cells were transduced twice to increase shRNA copy number and stableclones were established after selection in 80 μg/ml puromycin for 10days. Knock-down efficiency was determined by qPCR.

Mouse Xenograft Tumor Model

Female athymic, 18-20 g nude mice (nu/nu) were obtained from CharlesRiver (Willimantic, Conn.) and housed in microisolator cages, withautoclaved bedding in a specific pathogen-free facility, with a 12-hlight/dark cycle. Animals received water and food ad libitum, and wereobserved for signs of tumor growth, activity, feeding and pain, inaccordance with the study protocol approved by the VACHS IACUC.

Xenograft tumors were established by subcutaneous injection of BxPC3cells (2×10⁶ in 100 μl of PBS, pH 7.6). When the tumors reached a volumeof 50-100 mm³, the mice were divided a control group (n=14 treated withrabbit IgG, 40 μg by intraperitoneal injection (IP)), and anexperimental group (n=14) that received m28-RNLS (40 μg IP, every 3days). Tumor size was measured with digital calipers and volume wascalculated according to the formula (length× width)×π/2. In anothergroup of animals (n=6 each) sh-RNLS or sh-Control Panc1 cells (2×10⁶ in100 μl of PBS, pH 7.6) were injected subcutaneously. These animalsreceived no further treatments, and tumor size and volume were measuredfor up to 30 days.

At the end of the study, the mice were sacrificed, the tumors wereexcised and immediately snap-frozen in liquid nitrogen and stored at−80° C. Apoptosis was examined using the TUNEL assay (Roche in situApoptosis Detection System), according to the manufacturer'sinstructions. Sections were examined by light microscopy and theapoptosis index was determined by counting ≧1000 cells in 5 randomlyselected high-power fields (×200 magnification).

Statistical Analyses

The Wilcoxon rank test and the Mann-Whitney test were used for pairedand unpaired data, respectively. When appropriate, nonparametricrepeated-measures ANOVA (Friedman test) was used to evaluate statisticalsignificance. When the Friedman test revealed statistical significance,Dunn's test was used for pairwise comparisons. All data aremean±standard error of the mean (mean±SEM), and values of P<0.05 wereaccepted as a statistically significant difference. Statistical analysesof tissue array data were performed using SPSS® software, version 21.0(SPSS Inc., Chicago, Ill., USA). The results of this example are nowdescribed.

RNLS Overexpression in PDAC and Association with Decreased Survival

To determine if RNLS expression differed between normal and cancertissue, fifteen different types of cancer were examined by screeningcommercially available human tissue cDNA arrays using quantitative PCR(qPCR). RNLS expression was significantly increased in cancers of thepancreas, bladder and breast and in melanoma (FIG. 23A). Because oftheir particularly poor survival and limited therapeutic options, thefocus was on pancreatic neoplasms. RNLS expression was elevated in bothPDAC (˜3 fold) and pancreatic neuroendocrine (8 fold) tumors (FIG. 23B).Immunocytochemical studies using the anti-RNLS monoclonal m28-RNLSshowed that RNLS expression was present in PDAC grade 1-4 and waspredominantly localized to cancer cells, as shown in FIGS. 23C and 28).Most RNLS appeared to have a cytoplasmic distribution in cancer cells;it was present in all tumor grades, but was most evident inmore-differentiated cancers (Grades In neuroendocrine tumors of thepancreas, RNLS was expressed in cells throughout the tumor (FIG. 29).RNLS gene expression was greater in pancreatic ductal adenocarcinomacell (PDACC) lines with KRAS mutations (MiaPaCa2 and Panc1) than thosewith wild type KRAS, such as BxPC3 (FIG. 30).

RNLS expression in 69 patients with PDAC was characterized using tissuemicroarrays (TMA) consisting of formalin-fixed, paraffin-embedded tumorcores with matched adjacent normal tissue. The demographics and clinicalcharacteristics of the individuals from whom the samples were obtainedare shown in Table 3. Examination of 138 histo-spots from paired PDACtumors and their non-tumor adjacent tissues for RNLS protein expression,using an unbiased, quantitative, automated immunofluorescence microscopysystem (AQUA) (Gould Rothberg et al., 2009 Journal of Clinical Oncology.27(34):5772-80), showed that overall RNLS levels were more than 2-foldgreater in PDAC tumors than in their adjacent non-tumor pancreatictissue (p<0.001, FIG. 23D).

TABLE 3 Characteristics of patient cohort with PDAC CharacteristicsNumber/Total number Gender Female 24/69 (34.8%) Male 45/69 (65.2%) Age(years) Median 61 (36-85) 36-50 14/69 (20.3%) 51-69 39/69 (56.5%) 70-8516/69 (23.2%) Tumor Grade 1 1/69 (1.4%) 2 48/69 (69.6%) 3 15/69 (21.7%)4 1/69 (1.4%) Unknown 4/69 (5.8%) Survival (Months)  0-12 29/69 (42%)13-24 9/69 (13%) 25-48 18/69 (26%) 49-87 13/69 (19%)

To determine whether enhanced RNLS expression might affect PDAC'sclinical behavior, the question was asked whether the level ofexpression affected prognosis. Individuals whose tumors expressed highRNLS levels (n=34 with RNLS AQUA score >median) had a dramaticallyreduced 3-year survival rate (24% versus 49%, p=0.024, FIG. 23E). Thesefindings indicate that tumor levels of RNLS expression may be usefulprognostic markers in PDAC, and help identify a subset of patients witha more aggressive phenotype.

RNLS Signals Through PMCA4b and Functions as a Survival Factor forPancreatic Cancer Cells

RNLS-mediated signaling protects HK-2 cells exposed to toxic stress fromapoptosis (Lee et al., 2013 J Am Soc Nephrol. 24(3):445-55; Wang et al.,2014 Journal of the American Society of Nephrology.DOI:10.1681/asn.2013060665). To explore if RNLS signaling provided asurvival advantage to pancreatic ductal adenocarcinoma cells (PDACC)exposed to stress, serum was withdrawn from cultured BxPC3, Panc1, andMiaPaCa2 cells for 48 hours, and either recombinant RNLS (rRNLS) orbovine serum albumin (BSA) was added to the culture medium for anadditional 72 hrs; total and live (trypan blue exclusion) cell countswere determined. Compared to BSA, rRNLS increased PDACC survival rate by2 to 5 fold (FIG. 24A).

It has been shown that the cytoprotection afforded by the addition ofrRNLS to HK-2 cells exposed to hydrogen peroxide or cisplatin injury wasdependent on ERK activation (Lee et al., 2013 J Am Soc Nephrol.24(3):445-55; Wang et al., 2014 Journal of the American Society ofNephrology. DOI: 10.1681/asn.2013060665). The results shown in FIG. 24Bindicate that rRNLS also improves PDACC survival in an ERK-dependentmanner since pretreating with U0126 an inhibitor of the MAPK kinase MEK1abrogated rRNLS' protective effect.

Evidence regarding PMCA4b's role in RNLS dependent signaling inpancreatic cancer was obtained by specifically down-regulating PMCA4bexpression using siRNA. In control studies, non-targeting siRNAsaffected neither PMCA4b gene expression nor RNLS-mediated ERKphosphorylation (FIG. 24C). In contrast, PMCA4b-targeting siRNAsdecreased gene expression by more than 90%, and reduced RNLS dependentERK phosphorylation by ˜70% (FIG. 24C). PMCA4b inhibition had nodiscernable effect on RNLS mediated STAT3 phosphorylation suggesting theexistence of an additional RNLS receptor(s).

The observed increase in PDAC cell number in the presence of rRNLS isconsistent with RNLS signaling either preventing cell death and/orincreasing cell proliferation. The effect of RNLS on cell cycle wasexamined by fluorescence activated cell sorting (FACS) analysis todetermine if the apparent increase in PDACC viability was due toincreased cell proliferation or to a decrease in the rate of cell death.As shown in FIG. 24D, compared to treatment with BSA, rRNLS had noeffect on cell cycle progression, indicating that RNLS does not affectproliferation programs, but rather prevents cells death, and functionsas a survival factor.

Inhibitors of RNLS Signaling Block Pancreatic Cancer Growth

To determine the functional consequences of inhibiting RNLS expressionand signaling in pancreatic cancer cells, the effect of decreasing RNLSexpression on cell viability in vitro was evaluated by RNLS knockdown bysiRNA. This treatment markedly reduced the viability of the PDACC linesPanc1 and MiaPaCa2 (FIGS. 25A and 31). Since the RNLS peptide RP-220mimics the protective effect and signaling properties of rRNLS, it hasbeen reasoned that it likely interacts with a critical region of thereceptor for extracellular RNLS, and that antibodies generated againstit could be inhibitory. From a panel of monoclonal antibodies generatedin rabbit against RP-220, two clones, m28-RNLS, m37-RNLS, were selectedbased on their high binding affinity (KD of 0.316 and 2.67 nMrespectively). The inhibitory effects of m28-RNLS, m37-RNLS and of acommercially available polyclonal (against a partial sequence of RP-220)on PDACC growth are shown by the representative examples depicted inFIGS. 25B and 25C. These studies in cultured cells suggest that RNLS canact through an autocrine/paracrine pathway to stimulate PDACC growth.

To determine if inhibition of RNLS signaling affected tumor growth invivo, shRNA was used to generate two stably transfected Panc1 celllines: one containing non-targeting shRNA (sh-Control), and another withRNLS-targeting shRNA (sh-RNLS). RNLS expression in sh-RNLS cells wasdecreased by more than 90%, as assessed by qPCR (FIG. 31). Surprisingly,inhibition of RNLS expression by RNLS-targeting shRNA resulted in amarked reduction in the expression of its receptor PMCA4b, suggestingRNLS and PMCA4b expression are co-regulated (FIG. 32). The transfectedcells were injected subcutaneously into athymic nude mice and tumor sizewas assessed over a 30 day period. The tumor volume generated by sh-RNLScells was significantly smaller than that of sh-Control cells from day 8until day 30 when the animals were sacrificed (FIG. 25D). Since RNLSproduction and secretion by the host mouse were unaffected, theseresults indicate that sh-RNLS tumor cells were unresponsive tocirculating RNLS because of the concomitant inhibition the RNLS receptorPMCA4b.

To evaluate the therapeutic potential of inhibitory antibodies, BxPC3cells were subcutaneously injected into athymic, nude mice, which weretreated with either control rabbit IgG, or m28-RNLS, and tumor volumewas measured for up to 3 weeks. As shown in FIG. 25E, compared to rabbitIgG, m28-RNLS treatment caused a significant decrease in tumor volume.Together these studies in cultured PDACC cells and in an in vivo modelof PDACC provide compelling evidence that the RNLS pathway modulatespancreatic cancer growth and might serve as a therapeutic target.

Induction of Apoptosis and Cell Cycle Arrest in Tumor Cells by m28-RNLS

Sections of BxPC3 xenografted tumors from mice treated with eitherrabbit IgG or m28-RNLS to reduce RNLS levels revealed a ˜2-fold increasein apoptosis (TUNEL staining) (FIG. 26A) in the antibody-treated tumors:m28-RNLS vs IgG; 28.4±3.3 positive cells/high power field vs.IgG-14.8±2.3, n=14, p=0.002. FACS analysis of Panc1 cells in cultureconfirmed m28-RNLS caused apoptosis (FIGS. 26B and 33). Treatment withm28-RNLS antibody caused sustained phosphorylation of p38 MAPK beginningat day 1 post treatment (FIG. 26C).

The m28-RNLS treatment of BxPC3 tumors also led to a 2.5-fold decreasein the expression of a cellular proliferation marker Ki67 (m28-RNLS vsIgG: IgG, 137.1±14.9 vs 340.2±11.9 positive cells/high power field,n=14, p=1.4×10⁻⁸) (FIG. 26D, top panel), and to a ˜4-fold increase inthe expression of the cell cycle regulator p21 expression (m28-RNLS vsIgG: IgG, 178.1±11.4 vs 42.2±4.7.6 positive cells/high power field,n=14,)p=1.6×10⁻¹⁰ (FIG. 26D, bottom panel). FACS analysis of Panc1 cellswas performed to examine the effect of RNLS signaling inhibition on thecell cycle. The data shown in FIG. 26E confirm that RNLS inhibitioncaused apoptosis, as evidenced by the appearance of a large pre-G1 peak.They also reveal a marked decrease in G2 indicating that inhibition ofRNLS signaling by m28-RNLS causes a pre-G2 cell cycle arrest.

Presence of a Positive RNLS-STAT3 Feedback Loop and its Interruption bym28-RNLS

STAT3 binds to the promoter region of the RNLS gene and increases itsexpression (Sonawane et al., 2014 Biochemistry. 53(44):6878-6892). Apositive RNLS-STAT3 feedback loop is suggested by the observation thatin HK-2 cells treated with RNLS, STAT3 phosphorylation at serine 727(p-Ser⁷²⁷-STAT3) and tyrosine 705 (p-Y⁷⁰⁵-STAT3) increases 2 and 4 foldrespectively, but STAT1 is unaffected (FIG. 34). As depicted in FIGS.27A-B, the addition of RNLS to the PDACC line Panc1 caused a rapidincrease in phosphorylated STAT3 (p-Ser⁷²⁷-STAT3 and p-Y⁷⁰⁵-STAT3).Additional support for a RNLS-STAT3 feedback loop is provided by thefinding that inhibition of RNLS signaling in Panc1 by m28-RNLS leads toa long-lasting and sustained decrease in p-Y⁷⁰⁵-STAT3 (FIG. 27C-D).

SEQUENCES <SEQ ID NO: 1-antigenseq1a;PRT;homo sapiens> AVWDKADDSGGRMTTAC<SEQ ID NO: 2-antigenseq1b;PRT;homo sapiens> AVWDKAEDSGGRMTTAC<SEQ ID NO: 3-antigenseq1c;PRT;homo sapiens> CTPHYAKKHQRFYDEL<SEQ ID NO: 4-antigenseq1d;PRT;homo sapiens> CIRFVSIDNKKRNIESSEIGP<SEQ ID NO: 5-antigenseq1e;PRT;homo sapiens> PGQMTLHHKPFLAC<SEQ ID NO: 6-antigenseq1f;PRT;homo sapiens> CVLEALKNYI<SEQ ID NO: 7-antigenseq3a;PRT;homo sapiens> PSAGVILGC<SEQ ID NO: 8-HuRenalase-1 protein(polymorphismresulting in the glutamate amino acid at position 37);PRT;homo sapiens>MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKAEDSGGRMTTACSPHNPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKEGDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGSPEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFYEAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPFGVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVTNAAANCPGQMTLHHKPFLACGGDGFTQSNFDGCITSALCVLEALKNYI<SEQ ID NO: 9-1D-28-4 full length heavy chain amino acid;PRT;oryctolagus cuniculus>METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSFAVGWVRQAPGKGLEYIGIISSVGITRYASWAAGRFTISKTSTTVDLKITSPTTEDTATYFCARYGYSGDVNRLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMH EALHNHYTQKSISRSPGK<SEQ ID NO: 10-1D-28-4 full length light chainamino acid;PRT;oryctolagus cuniculus>MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQASQSVYDNNNLAWYQQKPGQPPKQLIYGASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGEFSCSSADCFAFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC<SEQ ID NO: 11-1D-28-4 heavy chain CDR1 aminoacid;PRT;oryctolagus cuniculus> LSSFAVG<SEQ ID NO: 12-1D-28-4 heavy chain CDR2 aminoacid;PRT;oryctolagus cuniculus> ITSSVGITRYASWAAG<SEQ ID NO: 13-1D-28-4 heavy chain CDR3 aminoacid;PRT;oryctolagus cuniculus> YGYSGDVNRLDL<SEQ ID NO: 14-1D-28-4 light chain CDR1 aminoacid;PRT;oryctolagus cuniculus> SQSVYDNNNLA<SEQ ID NO: 15-1D-28-4 light chain CDR2 aminoacid;PRT;oryctolagus cuniculus> GASTLAS<SEQ ID NO: 16-1D-28-4 light chain CDR3 aminoacid;PRT;oryctolagus cuniculus> LGEFSCSSADCFA<SEQ ID NO: 17-1D-37-10 full length heavy chainamino acid;PRT;oryctolagus cuniculus>METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGGSLTLTCTVSGFSLSDYAIIWVRQAPGKGLEYIAIIGSSGDTFYATWAKGRFTISKTSTTVDLKMTSLTAADTATYFCAPRYAGTTDYHDAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSV MHEALHNHYTQKSISRSPGK<SEQ ID NO: 18-1D-37-10 full length light chainamino acid;PRT;oryctolagus cuniculus>MDTRAPTQLLGLLLLWLPGARCAEVVMTQTPASMEAPMGGTVTIKCQASQNIYNYLSWYQQKPGQPPKLLVYKASTLTSGVPSRFKGSGSGTQFTLTISDLECADAATYYCQINYSIYNHYNIIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC<SEQ ID NO: 19-1D-37-10 heavy chain CDR1 aminoacid;PRT;oryctolagus cuniculus> LSDYAII<SEQ ID NO: 20-1D-37-10 heavy chain CDR2 aminoacid;PRT;oryctolagus cuniculus> IIGSSGDTFYATWAKG<SEQ ID NO: 21-1D-37-10 heavy chain CDR3 aminoacid;PRT;oryctolagus cuniculus> RYAGTTDYHDAFDP<SEQ ID NO: 22-1D-37-10 light chain CDR1 aminoacid;PRT;oryctolagus cuniculus> SQNIYNYLS<SEQ ID NO: 23-1D-37-10 light chain CDR2 aminoacid;PRT;oryctolagus cuniculus> KASTLTS<SEQ ID NO: 24-1D-37-10 light chain CDR3 aminoacid;PRT;oryctolagus cuniculus> QINYSIYNHYNII<SEQ ID NO: 25-1F-26-1 full length heavy chainamino acid;PRT;oryctolagus cuniculus>METGLRWLLLVAVLKGVQCQSVKESEGGLFKPTDTLTLTCTVSGFSLSSYGVTWVRQAPGNGLEWIGLIGDRGTTFYASWAKSRSTITRNTNLNTVTLKMTRLTAADTATYFCARGSGYGARIWGPGTLVTVSSWQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEA LHNHYTQKSISRSPGK<SEQ ID NO: 26-1F-26-1 full length light chain amino acid;PRT;oryctolagus cuniculus>MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQSSQSVYKNNYLAWYQQKPGQPPKLLIYETSKLASGVPPRFSGSGSGTQFTLTISSVQCDDAATYYCQGGYSGVDFMAFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC<SEQ ID NO: 27-1F-26-1 heavy chain CDR1 aminoacid;PRT;oryctolagus cuniculus> LSSYGVT<SEQ ID NO: 28-1F-26-1 heavy chain CDR2 aminoacid;PRT;oryctolagus cuniculus> LIGDRGTTFYASWAKS<SEQ ID NO: 29-1F-26-1 heavy chain CDR3 amino acid;PRT;oryctolagus cuniculus> GSGYGARI<SEQ ID NO: 30-1F-26-1 light chain CDR1 aminoacid;PRT;oryctolagus cuniculus> SQSVYKNNYLA<SEQ ID NO: 31-1F-26-1 light chain CDR2 aminoacid;PRT;oryctolagus cuniculus> ETSKLAS<SEQ ID NO: 32-1F-26-1 light chain CDR3 aminoacid;PRT;oryctolagus cuniculus> QGGYSGVDFMA<SEQ ID NO: 33-1F-42-7 full length heavy chainamino acid;PRT;oryctolagus cuniculus>METGLRWLLLVAVLKGVQCQSVKESEGGLFKPTDTLTLTCTVSGFSLTTYGVTWVRQAPGNGLEWIGLIGDRGTTYYASWVNGRSTITRNTNLNTVTLKMTRLTAADTATYFCARGSGYGARIWGPGTLVTVASWQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEA LHNHYTQKSISRSPGK<SEQ ID NO: 34-1F-42-7 full length light chainamino acid;PRT;oryctolagus cuniculus>MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPMSAALGGTVTINCQSSQTVYNNNYLSWYQQKPGQPPKLLIYETSKLSSGVPPRFSGSGSGTQFTLTISSVQCDDAATYYCQGGYSGVDFMAFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC<SEQ ID NO: 35-1F-42-7 heavy chain CDR1 aminoacid;PRT;oryctolagus cuniculus> LTTYGVT<SEQ ID NO: 36-1F-42-7 heavy chain CDR2 aminoacid;PRT;oryctolagus cuniculus> LIGDRGTTYYASWVNG<SEQ ID NO: 37-1F-42-7 heavy chain CDR3 aminoacid;PRT;oryctolagus cuniculus> GSGYGARI<SEQ ID NO: 38-1F-42-7 light chain CDR1 aminoacid;PRT;oryctolagus cuniculus> SQTVYNNNYLS<SEQ ID NO: 39-1F-42-7 light chain CDR2 aminoacid;PRT;oryctolagus cuniculus> ETSKLSS<SEQ ID NO: 40-1F-42-7 light chain CDR3 aminoacid;PRT;oryctolagus cuniculus> QGGYSGVDFM<SEQ ID NO: 41-3A-5-2 full length heavy chainamino acid;PRT;oryctolagus cuniculus>METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLNNYHIYWVRQAPGKGLEYIGIIFNGGTYYARWTKGRFTISKTSTTVDLKMTSLTTEDTATYFCARGDGIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQ KSISRSPGK<SEQ ID NO: 42-3A-5-2 full length light chainamino acid;PRT;oryctolagus cuniculus>MDTRAPTQLLGLLLLWLPGATFAQVLTQTPASVSAAVGGTVTINCQASQSVFNNNYLAWYQQKPGQPPKRLIYSASTLASGVSSRFKGSGSGTEFTLTMSGVECDDAATYYCAGSFDCNSGDCVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC<SEQ ID NO: 43-3A-5-2 heavy chain CDR1 aminoacid;PRT;oryctolagus cuniculus> LNNYHIY<SEQ ID NO: 44-3A-5-2 heavy chain CDR2 aminoacid;PRT;oryctolagus cuniculus> IIFNGGTYYARWTKG<SEQ ID NO: 45-3A-5-2 heavy chain CDR3 aminoacid;PRT;oryctolagus cuniculus> GDGI<SEQ ID NO: 46-3A-5-2 light chain CDR1 aminoacid;PRT;oryctolagus cuniculus> SQSVFNNNYLA<SEQ ID NO: 47-3A-5-2 light chain CDR2 aminoacid;PRT;oryctolagus cuniculus> SASTLAS<SEQ ID NO: 48-3A-5-2 light chain CDR3 aminoacid;PRT;oryctolagus cuniculus> AGSFDCNSGDCVA<SEQ ID NO: 49-Human Renalase-1 nucleic acidsequence (possible polymorphism at nucleotideposition 111);DNA;homo sapiens>ATGGCGCAGGTGCTGATCGTGGGCGCCGGGATGACAGGAAGCTTGTGCGCTGCGCTGCTGAGGAGGCAGACGTCCGGTCCCTTGTACCTTGCTGTGTGGGACAAGGCTGAGGACTCAGGGGGAAGAATGACTACAGCCTGCAGTCCTCATAATCCTCAGTGCACAGCTGACTTGGGTGCTCAGTACATCACCTGCACTCCTCATTATGCCAAAAAACACCAACGTTTTTATGATGAACTGTTAGCCTATGGCGTTTTGAGGCCTCTAAGCTCGCCTATTGAAGGAATGGTGATGAAAGAAGGAGACTGTAACTTTGTGGCACCTCAAGGAATTTCTTCAATTATTAAGCATTACTTGAAAGAATCAGGTGCAGAAGTCTACTTCAGACATCGTGTGACACAGATCAACCTAAGAGATGACAAATGGGAAGTATCCAAACAAACAGGCTCCCCTGAGCAGTTTGATCTTATTGTTCTCACAATGCCAGTTCCTGAGATTCTGCAGCTTCAAGGTGACATCACCACCTTAATTAGTGAATGCCAAAGGCAGCAACTGGAGGCTGTGAGCTACTCCTCTCGATATGCTCTGGGCCTCTTTTATGAAGCTGGTACGAAGATTGATGTCCCTTGGGCTGGGCAGTACATCACCAGTAATCCCTGCATACGCTTCGTCTCCATTGATAATAAGAAGCGCAATATAGAGTCATCAGAAATTGGGCCTTCCCTCGTGATTCACACCACTGTCCCATTTGGAGTTACATACTTGGAACACAGCATTGAGGATGTGCAAGAGTTAGTCTTCCAGCAGCTGGAAAACATTTTGCCGGGTTTGCCTCAGCCAATTGCTACCAAATGCCAAAAATGGAGACATTCACAGGTTACAAATGCTGCTGCCAACTGTCCTGGCCAAATGACTCTGCATCACAAACCTTTCCTTGCATGTGGAGGGGATGGATTTACTCAGTCCAACTTTGATGGCTGCATCACTTCTGCCCTATGTGTTCTGGAAGCTTTAAAGAATTATATTTAA<SEQ ID NO: 50-Human Renalase-2 amino acid sequence (polymorphism resulting in theglutamate amino acid at position 37;PRT; homo sapiens>MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKAEDSGGRMTTACSPHNPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKEGDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGSPEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFYEAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPFGVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVP SAGVILGCAKSPWMMAIGFPI<SEQ ID NO: 51-Human Renalase-2 nucleic acidsequence (possible polymorphism atnucleotide position 111);DNA;homo sapiens>ATGGCGCAGGTGCTGATCGTGGGCGCCGGGATGACAGGAAGCTTGTGCGCTGCGCTGCTGAGGAGGCAGACGTCCGGTCCCTTGTACCTTGCTGTGTGGGACAAGGCTGAGGACTCAGGGGGAAGAATGACTACAGCCTGCAGTCCTCATAATCCTCAGTGCACAGCTGACTTGGGTGCTCAGTACATCACCTGCACTCCTCATTATGCCAAAAAACACCAACGTTTTTATGATGAACTGTTAGCCTATGGCGTTTTGAGGCCTCTAAGCTCGCCTATTGAAGGAATGGTGATGAAAGAAGGAGACTGTAACTTTGTGGCACCTCAAGGAATTTCTTCAATTATTAAGCATTACTTGAAAGAATCAGGTGCAGAAGTCTACTTCAGACATCGTGTGACACAGATCAACCTAAGAGATGACAAATGGGAAGTATCCAAACAAACAGGCTCCCCTGAGCAGTTTGATCTTATTGTTCTCACAATGCCAGTTCCTGAGATTCTGCAGCTTCAAGGTGACATCACCACCTTAATTAGTGAATGCCAAAGGCAGCAACTGGAGGCTGTGAGCTACTCCTCTCGATATGCTCTGGGCCTCTTTTATGAAGCTGGTACGAAGATTGATGTCCCTTGGGCTGGGCAGTACATCACCAGTAATCCCTGCATACGCTTCGTCTCCATTGATAATAAGAAGCGCAATATAGAGTCATCAGAAATTGGGCCTTCCCTCGTGATTCACACCACTGTCCCATTTGGAGTTACATACTTGGAACACAGCATTGAGGATGTGCAAGAGTTAGTCTTCCAGCAGCTGGAAAACATTTTGCCGGGTTTGCCTCAGCCAATTGCTACCAAATGCCAAAAATGGAGACATTCACAGGTACCAAGTGCTGGTGTGATTCTAGGATGTGCGAAGAGCCCCTGGATGATGGCGA TTGGATTTCCCATC<SEQ ID NO: 52-1D-28-4 full length heavy chain nucleic acid;DNA;oryctolagus cuniculus>ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGTTTTGCAGTGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAGTAGTGTTGGTATTACACGCTACGCGAGCTGGGCGGCCGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTTTGTGCCAGATATGGTTATAGTGGTGATGTTAATCGGTTGGATCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGG GTAAATGA<SEQ ID NO: 53-1D-28-4 full length light chain nucleic acid;DNA;oryctolagus cuniculus>ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTAGTAGTGCTGATTGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG<SEQ ID NO: 54-1D-28-4 heavy chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> ctcagtagttttgcagtgggc<SEQ ID NO: 55-1D-28-4 heavy chain CDR2 nucleicacid;DNA;oryctolagus cuniculus>atcattagtagtgttggtattacacgctacgcgagctgggcggccggc<SEQ ID NO: 56-1D-28-4 heavy chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> tatggttatagtggtgatgttaatcggttggatctc<SEQ ID NO: 57-1D-28-4 light chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> agtcagagtgtttatgataacaacaacttagcc<SEQ ID NO: 58-1D-28-4 light chain CDR2 nucleicacid;DNA;oryctolagus cuniculus> ggtgcatccactctggcatct<SEQ ID NO: 59-1D-28-4 light chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> ctaggcgaatttagttgtagtagtgctgattgttttgct<SEQ ID NO: 60-1D-37-10 full length heavy chainnucleic acid;DNA;oryctolagus cuniculus>ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTGACTATGCAATAATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGCAATTATTGGTAGTAGTGGTGACACATTCTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCCCCACGTTATGCTGGTACTACTGATTATCATGATGCTTTTGATCCCTGGGGCCCAGGCACTTTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCT CTCCGGGTAAATGA<SEQ ID NO: 61-1D-37-10 full length light chainnucleic acid;DNA;oryctolagus cuniculus>ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCGAAGTAGTGATGACCCAGACTCCAGCCTCCATGGAGGCACCTATGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTTACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTAGTCTACAAGGCCTCCACTCTGACTTCTGGGGTCCCGTCGCGCTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAATCAATTACTCTATTTATAATCATTATAATATTATTTTTGGCGGAGGGACCGAGGTGGTCGTCAAGGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG<SEQ ID NO: 62-1D-37-10 heavy chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> ctcagtgactatgcaataatc<SEQ ID NO: 63-1D-37-10 heavy chain CDR2 nucleicacid;DNA;oryctolagus cuniculus>attattggtagtagtggtgacacattctacgcgacctgggcgaaaggc<SEQ ID NO: 64-1D-37-10 heavy chain CDR3 nucleicacid;DNA;oryctolagus cuniculus>cgttatgctggtactactgattatcatgatgcttttgatccc<SEQ ID NO: 65-1D-37-10 light chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> agtcagaacatttacaactacttatcc<SEQ ID NO: 66-1D-37-10 light chain CDR2 nucleicacid;DNA;oryctolagus cuniculus> aaggcctccactctgacttct<SEQ ID NO: 67-1D-37-10 light chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> caaatcaattactctatttataatcattataatattatt<SEQ ID NO: 68-1F-26-1 full length heavy chainnucleic acid;DNA;oryctolagus cuniculus>ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAGTGGATCGGATTGATTGGTGATCGTGGTACTACGTTCTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGGCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGGGGAGTGGGTATGGTGCTCGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCATGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAAT GA<SEQ ID NO: 69-1F-26-1 full length light chainnucleic acid;DNA;oryctolagus cuniculus>ATGGACACGAGGGCCCCCACTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCGCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTTATCTACGAAACATCCAAACTGGCATCTGGGGTCCCACCGCGGTTCAGCGGCAGTGGGTCTGGGACACAGTTCACTCTCACCATCAGCAGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGGTTATAGTGGTGTTGATTTTATGGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG<SEQ ID NO: 70-1F-26-1 heavy chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> ctcagtagctatggagtgacc<SEQ ID NO: 71-1F-26-1 heavy chain CDR2 nucleicacid;DNA;oryctolagus cuniculus>ttgattggtgatcgtggtactacgttctacgcgagctgggcgaaaagc<SEQ ID NO: 72-1F-26-1 heavy chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> Gggagtgggtatggtgctcgcatc<SEQ ID NO: 73-1F-26-1 light chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> agtcagagtgtttataagaacaactacttagcc<SEQ ID NO: 74-1F-26-1 light chain CDR2 nucleicacid;DNA;oryctolagus cuniculus> gaaacatccaaactggcatct<SEQ ID NO: 75-1F-26-1 light chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> caaggcggttatagtggtgttgattttatggct<SEQ ID NO: 76-1F-42-7 full length heavy chainnucleic acid;DNA;oryctolagus cuniculus>ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCACTACCTATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAATGGGCTGGAGTGGATCGGATTGATTGGTGATCGCGGTACCACTTACTACGCGAGCTGGGTGAATGGCCGATCCACCATCACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGGCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGGGGAGTGGATATGGTGCTCGCATCTGGGGCCCAGGCACCCTGGTCACCGTCGCCTCATGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAAT GA<SEQ ID NO: 77-1F-42-7 full length light chain nucleic acid;DNA;oryctolagus cuniculus>ATGGACACGAGGGCCCCCACTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCCATGTCTGCAGCTCTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGACTGTTTATAACAATAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTTATCTACGAAACATCCAAACTGTCATCTGGGGTCCCACCGCGGTTCAGCGGCAGTGGGTCTGGGACACAGTTCACTCTCACCATCAGCAGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGGTTATAGTGGTGTTGATTTTATGGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG<SEQ ID NO: 78-1F-42-7 heavy chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> ctcactacctatggagtgacc<SEQ ID NO: 79-1F-42-7 heavy chain CDR2 nucleicacid;DNA;oryctolagus cuniculus>ttgattggtgatcgcggtaccacttactacgcgagctgggtgaatggc<SEQ ID NO: 80-1F-42-7 heavy chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> gggagtggatatggtgctcgcatc<SEQ ID NO: 81-1F-42-7 light chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> agtcagactgtttataacaataactacttatcc<SEQ ID NO: 82-1F-42-7 light chain CDR2 nucleicacid;DNA;oryctolagus cuniculus> gaaacatccaaactgtcatct<SEQ ID NO: 83-1F-42-7 light chain CDR3 nucleic acid;DNA;oryctolagus cuniculus> ggcggttatagtggtgttgattttatggct<SEQ ID NO: 84-3A-5-2 full length heavy chainnucleic acid;DNA;oryctolagus cuniculus>ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAATAACTACCACATATACTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGAATCATTTTCAATGGTGGCACATATTACGCGAGATGGACAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGGGACGGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA<SEQ ID NO: 85-3A-5-2 full length light chainnucleic acid;DNA;oryctolagus cuniculus>ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTTTAATAACAACTATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATTCTGCATCCACTCTGGCGTCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAATTCACTCTGACCATGAGTGGCGTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCAGTTTTGATTGTAATAGTGGTGATTGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAGGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG<SEQ ID NO: 86-3A-5-2 heavy chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> ctcaataactaccacatatac<SEQ ID NO: 87-3A-5-2 heavy chain CDR2 nucleicacid;DNA;oryctolagus cuniculus>atcattttcaatggtggcacatattacgcgagatggacaaaaggc<SEQ ID NO: 88-3A-5-2 heavy chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> ggggacggcatc<SEQ ID NO: 89-3A-5-2 light chain CDR1 nucleicacid;DNA;oryctolagus cuniculus> agtcagagtgtttttaataacaactatttagcc<SEQ ID NO: 90-3A-5-2 light chain CDR2 nucleicacid;DNA;oryctolagus cuniculus> tctgcatccactctggcgtct<SEQ ID NO: 91-3A-5-2 light chain CDR3 nucleicacid;DNA;oryctolagus cuniculus> Gcaggcagttttgattgtaatagtggtgattgtgttgct<SEQ ID NO: 92-alternative Human Renalase-1 protein (polymorphism resulting in theaspartate amino acid at position 37;PRT; homo sapiens>MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKADDSGGRMTTACSPHNPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKEGDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGSPEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFYEAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPFGVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVTNAAANCPGQMTLHHKPFLACGGDGFTQSNFDGCITSALCVLEALKNYI<SEQ ID NO: 93-alternative Human Renalase-1nucleic acid sequence (note possiblepolymorphism at nucleotide position 111; DNA;homo sapiens>ATGGCGCAGGTGCTGATCGTGGGCGCCGGGATGACAGGAAGCTTGTGCGCTGCGCTGCTGACGAGGCAGACGTCCGGTCCCTTGTACCTTGCTGTGTGGGACAAGGCTGAGGACTCAGGGGGAAGAATGACTACAGCCTGCAGTCCTCATAATCCTCAGTGCACAGCTGACTTGGGTGCTCAGTACATCACCTGCACTCCTCATTATGCCAAAAAACACCAACGTTTTTATGATGAACTGTTAGCCTATGGCGTTTTGAGGCCTCTAAGCTCGCCTATTGAAGGAATGGTGATGAAAGAAGGAGACTGTAACTTTGTGGCACCTCAAGGAATTTCTTCAATTATTAAGCATTACTTGAAAGAATCAGGTGCAGAAGTCTACTTCAGACATCGTGTGACACAGATCAACCTAAGAGATGACAAATGGGAAGTATCCAAACAAACAGGCTCCCCTGAGCAGTTTGATCTTATTGTTCTCACAATGCCAGTTCCTGAGATTCTGCAGCTTCAAGGTGACATCACCACCTTAATTAGTGAATGCCAAAGGCAGCAACTGGAGGCTGTGAGCTACTCCTCTCGATATGCTCTGGGCCTCTTTTATGAAGCTGGTACGAAGATTGATGTCCCTTGGGCTGGGCAGTACATCACCAGTAATCCCTGCATACGCTTCGTCTCCATTGATAATAAGAAGCGCAATATAGAGTCATCAGAAATTGGGCCTTCCCTCGTGATTCACACCACTGTCCCATTTGGAGTTACATACTTGGAACACAGCATTGAGGATGTGCAAGAGTTAGTCTTCCAGCAGCTGGAAAACATTTTGCCGGGTTTGCCTCAGCCAATTGCTACCAAATGCCAAAAATGGAGACATTCACAGGTTACAAATGCTGCTGCCAACTGTCCTGGCCAAATGACTCTGCATCACAAACCTTTCCTTGCATGTGGAGGGGATGGATTTACTCAGTCCAACTTTGATGGCTGCATCACTTCTGCCCTATGTGTTCTGGAAGCTTTAAAGAATTATATTTAA<SEQ ID NO: 94-alternative Human Renalase-2 aminoacid sequence (polymorphism resulting in theaspartate amino acid at position 37;PRT; homo sapiens>MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKADDSGGRMTTACSPHNPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKEGDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGSPEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFYEAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPFGVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVP SAGVILGCAKSPWMMAIGFPI<SEQ ID NO: 95-alternative Human Renalase-2nucleic acid sequence (note possiblepolymorphism at nucleotide position 111; DNA;homo sapiens>ATGGCGCAGGTGCTGATCGTGGGCGCCGGGATGACAGGAAGCTTGTGCGCTGCGCTGCTGACGAGGCAGACGTCCGGTCCCTTGTACCTTGCTGTGTGGGACAAGGCTGAGGACTCAGGGGGAAGAATGACTACAGCCTGCAGTCCTCATAATCCTCAGTGCACAGCTGACTTGGGTGCTCAGTACATCACCTGCACTCCTCATTATGCCAAAAAACACCAACGTTTTTATGATGAACTGTTAGCCTATGGCGTTTTGAGGCCTCTAAGCTCGCCTATTGAAGGAATGGTGATGAAAGAAGGAGACTGTAACTTTGTGGCACCTCAAGGAATTTCTTCAATTATTAAGCATTACTTGAAAGAATCAGGTGCAGAAGTCTACTTCAGACATCGTGTGACACAGATCAACCTAAGAGATGACAAATGGGAAGTATCCAAACAAACAGGCTCCCCTGAGCAGTTTGATCTTATTGTTCTCACAATGCCAGTTCCTGAGATTCTGCAGCTTCAAGGTGACATCACCACCTTAATTAGTGAATGCCAAAGGCAGCAACTGGAGGCTGTGAGCTACTCCTCTCGATATGCTCTGGGCCTCTTTTATGAAGCTGGTACGAAGATTGATGTCCCTTGGGCTGGGCAGTACATCACCAGTAATCCCTGCATACGCTTCGTCTCCATTGATAATAAGAAGCGCAATATAGAGTCATCAGAAATTGGGCCTTCCCTCGTGATTCACACCACTGTCCCATTTGGAGTTACATACTTGGAACACAGCATTGAGGATGTGCAAGAGTTAGTCTTCCAGCAGCTGGAAAACATTTTGCCGGGTTTGCCTCAGCCAATTGCTACCAAATGCCAAAAATGGAGACATTCACAGGTACCAAGTGCTGGTGTGATTCTAGGATGTGCGAAGAGCCCCTGGATGATGGCGA TTGGATTTCCCATC

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A composition comprising at least one renalase inhibitor selectedfrom the group consisting of a chemical compound, a protein, a peptide,a peptidomemetic, a renalase receptor, a renalase receptor fragment, anantibody, an antibody fragment, an antibody mimetic, a ribozyme, a smallmolecule chemical compound, an short hairpin RNA, an antisense nucleicacid molecule, siRNA, miRNA, a nucleic acid encoding an antisensenucleic acid molecule, a nucleic acid sequence encoding a protein. 2.The composition of claim 1, wherein the renalase inhibitor is a renalasebinding molecule.
 3. The composition of claim 2, wherein the renalasebinding molecule is an antibody or binding portion thereof.
 4. Thecomposition of claim 3, comprising an antibody or binding portionthereof that specifically binds to renalase with an affinity of at least10⁻⁶ M.
 5. The composition of claim 3, wherein the antibody specificallybinds a peptide sequence selected from the group consisting of SEQ IDNO: 1-7.
 6. (canceled)
 7. The composition of claim 3, wherein theantibody is selected from the group consisting of a monoclonal antibody,a polyclonal antibody, a single chain antibody, an immunoconjugate, adefucosylated antibody, and a bispecific antibody.
 8. The composition ofclaim 7, wherein the immunoconjugate comprises a therapeutic agent or adetection moiety.
 9. The composition of claim 3, wherein the antibody isselected from the group consisting of a humanized antibody, a chimericantibody, a fully human antibody, an antibody mimetic.
 10. Thecomposition of claim 3, wherein the antibody comprises at least oneselected from the group consisting of: a) the heavy chain CDR1 sequenceselected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 19;b) the heavy chain CDR2 sequence selected from the group consisting ofSEQ ID NO: 12 and SEQ ID NO: 20; c) the heavy chain CDR3 sequenceselected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 21;d) the light chain CDR1 sequence selected from the group consisting ofSEQ ID NO: 14 and SEQ ID NO: 22; e) the light chain CDR2 sequenceselected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 23;f) the light chain CDR3 sequence selected from the group consisting ofSEQ ID NO: 16 and SEQ ID NO:
 24. 11. The composition of claim 4, whereinthe antibody specifically binds a polypeptide comprising the amino acidsequence of SEQ ID NO:
 4. 12. The composition of claim 3, wherein theantibody comprises at least one selected from the group consisting of:a) the heavy chain CDR1 sequence selected from the group consisting ofSEQ ID NO: 27 and SEQ ID NO: 35; b) the heavy chain CDR2 sequenceselected from the group consisting of SEQ ID NO: 28 and SEQ ID NO: 36;c) the heavy chain CDR3 sequence selected from the group consisting ofSEQ ID NO: 29 and SEQ ID NO: 37; d) the light chain CDR1 sequenceselected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 38;e) the light chain CDR2 sequence selected from the group consisting ofSEQ ID NO: 31 and SEQ ID NO: 39; f) the light chain CDR3 sequenceselected from the group consisting of SEQ ID NO: 32 and SEQ ID NO: 40.13. The composition of claim 6, wherein the antibody specifically bindsa polypeptide comprising the amino acid sequence of SEQ ID NO:
 6. 14.The composition of claim 3, wherein the antibody comprises at least oneselected from the group consisting of: a) the heavy chain CDR1 sequenceSEQ ID NO: 43; b) the heavy chain CDR2 sequence SEQ ID NO: 44; c) theheavy chain CDR3 sequence SEQ ID NO: 45; d) the light chain CDR1sequence SEQ ID NO: 46; e) the light chain CDR2 sequence SEQ ID NO: 47;f) the light chain CDR3 sequence SEQ ID NO:
 48. 15. The composition ofclaim 7, wherein the antibody specifically binds a polypeptidecomprising the amino acid sequence of SEQ ID NO:
 7. 16. The compositionof claim 3, wherein the antibody comprises a heavy chain sequenceselected from the group consisting of SEQ ID NOs: 9, 17, 25, 33, and 41.17. The composition of claim 3, wherein the antibody comprises a lightchain sequence selected from the group consisting of SEQ ID NOs: 10, 18,26, 34, and
 42. 18. A composition comprising an antibody that binds torenalase and competes with the binding of the antibody of claim 3 torenalase.
 19. A method of treating or preventing a disease or disorderassociated with renalase in a subject, the method comprising the step ofadministering to the subject the composition of claim
 1. 20. The methodof claim 19, wherein the composition is administered to the subject incombination with a second therapeutic agent.
 21. The method of claim 19,wherein the disease or disorder associated with renalase is selectedfrom the group consisting of renal disease, cardiovascular disease,cancer, and any combination thereof.
 22. The method of claim 21, whereinthe disease or disorder is cancer, and the cancer is pancreatic canceror melanoma. 23-36. (canceled)